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Sample records for controlling eukaryotic cell

  1. Growth control of the eukaryote cell: a systems biology study in yeast

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    Castrillo Juan I

    2007-04-01

    Full Text Available Abstract Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for

  2. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

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    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.

  3. Endosymbiosis and Eukaryotic Cell Evolution.

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    Archibald, John M

    2015-10-05

    Understanding the evolution of eukaryotic cellular complexity is one of the grand challenges of modern biology. It has now been firmly established that mitochondria and plastids, the classical membrane-bound organelles of eukaryotic cells, evolved from bacteria by endosymbiosis. In the case of mitochondria, evidence points very clearly to an endosymbiont of α-proteobacterial ancestry. The precise nature of the host cell that partnered with this endosymbiont is, however, very much an open question. And while the host for the cyanobacterial progenitor of the plastid was undoubtedly a fully-fledged eukaryote, how - and how often - plastids moved from one eukaryote to another during algal diversification is vigorously debated. In this article I frame modern views on endosymbiotic theory in a historical context, highlighting the transformative role DNA sequencing played in solving early problems in eukaryotic cell evolution, and posing key unanswered questions emerging from the age of comparative genomics.

  4. Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote.

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    Miyagishima, Shin-ya; Fujiwara, Takayuki; Sumiya, Nobuko; Hirooka, Shunsuke; Nakano, Akihiko; Kabeya, Yukihiro; Nakamura, Mami

    2014-05-08

    Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain unknown. Here, using the unicellular red alga Cyanidioschyzon merolae, we show that the G1/S regulator RBR-E2F-DP complex links the G1/S transition to circadian rhythms. Time-dependent E2F phosphorylation promotes the G1/S transition during subjective night and this phosphorylation event occurs independently of cell cycle progression, even under continuous dark or when cytosolic translation is inhibited. Constitutive expression of a phospho-mimic of E2F or depletion of RBR unlinks cell cycle progression from circadian rhythms. These transgenic lines are exposed to higher oxidative stress than the wild type. Circadian inhibition of cell cycle progression during the daytime by RBR-E2F-DP pathway likely protects cells from photosynthetic oxidative stress by temporally compartmentalizing photosynthesis and cell cycle progression.

  5. Evolutionary origin of eukaryotic cells.

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    Kostianovsky, M

    2000-01-01

    This article reviews literature on the transition from rudimentary prokaryotic life to eukaryotes. An overview of the differences between these organisms and theories of eukaryogenesis are reviewed. Various methods of investigating the transformation from prokaryotes to eukaryotes are elaborated, including the fossil, the molecular and living records, and examples are given. Lastly, the recent molecular studies and the impact on phylogenetic classification for the tree of life, based on molecular evolution, are discussed.

  6. Is hypusine essential for eukaryotic cell proliferation?

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    Park, M H; Wolff, E C; Folk, J E

    1993-12-01

    Hypusine [N epsilon-(4-amino-2-hydroxybutyl)-L-lysine] is a most remarkable amino acid, occurring in all eukaryotic cells, yet occupying only a single position in one protein, eukaryotic protein synthesis initiation factor 5A (eIF-5A). The unusual structure of hypusine, its derivation from the polyamine spermidine, and its increased formation in response to growth stimulation, as well as its limited occurrence in the highly conserved amino acid sequence of eIF-5A, have aroused keen interest in the biological significance of its existence and in its relationship to eIF-5A function.

  7. Hypusine is essential for eukaryotic cell proliferation.

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    Park, M H; Lee, Y B; Joe, Y A

    1997-01-01

    Hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] occurs in all eukaryotes at one residue in a highly conserved protein, the putative eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D). This unusual amino acid is produced in a unique posttranslational modification reaction that involves the conjugation of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the deoxyhypusine [N epsilon-(4-aminobutyl)lysine] residue and its subsequent hydroxylation. The strict specificity of hypusine synthesis, its derivation from spermidine and its requirement for the activity of eIF-5A and for eukaryotic cell proliferation have raised keen interest in the physiological function of the hypusine-containing protein, eIF-5A.

  8. Sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus.

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    Bell, Philip John Livingstone

    2006-11-07

    The origin of the eukaryotic cell cycle, including mitosis, meiosis, and sex are as yet unresolved aspects of the evolution of the eukaryotes. The wide phylogenetic distribution of both mitosis and meiosis suggest that these processes are integrally related to the origin of the earliest eukaryotic cells. According to the viral eukaryogenesis (VE) hypothesis, the eukaryotes are a composite of three phylogenetically unrelated organisms: a viral lysogen that evolved into the nucleus, an archaeal cell that evolved into the eukaryotic cytoplasm, and an alpha-proteobacterium that evolved into the mitochondria. In the extended VE hypothesis presented here, the eukaryotic cell cycle arises as a consequence of the derivation of the nucleus from a lysogenic DNA virus.

  9. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

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    Boitchenko, Veronika E.; Korobko, Vyacheslav G.; Prassolov, Vladimir S.; Kravchenko, Vladimir V.; Kuimov, Alexander N.; Turetskaya, Regina L.; Kuprash, Dmitry V.; Nedospasov, Sergei A.

    2000-10-01

    Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans. In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha. Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

  10. The size-wise nucleus: nuclear volume control in eukaryotes.

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    Huber, Michael D; Gerace, Larry

    2007-11-19

    Eukaryotic cells have an "awareness" of their volume and organellar volumes, and maintain a nuclear size that is proportional to the total cell size. New studies in budding and fission yeast have examined the relationship between cell and nuclear volumes. It was found that the size of the nucleus remains proportional to cell size in a wide range of genetic backgrounds and growth conditions that alter cell volume and DNA content. Moreover, in multinucleated fission yeast cells, Neumann and Nurse (see p. 593 of this issue) found that the sizes of individual nuclei are controlled by the relative amount of cytoplasm surrounding each nucleus. These results highlight a role of the cytoplasm in nuclear size control.

  11. Prevention of DNA re-replication in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Lan N. Truong; Xiaohua Wu

    2011-01-01

    DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability.

  12. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

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    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  13. An inside-out origin for the eukaryotic cell.

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    Baum, David A; Baum, Buzz

    2014-10-28

    Although the origin of the eukaryotic cell has long been recognized as the single most profound change in cellular organization during the evolution of life on earth, this transition remains poorly understood. Models have always assumed that the nucleus and endomembrane system evolved within the cytoplasm of a prokaryotic cell. Drawing on diverse aspects of cell biology and phylogenetic data, we invert the traditional interpretation of eukaryotic cell evolution. We propose that an ancestral prokaryotic cell, homologous to the modern-day nucleus, extruded membrane-bound blebs beyond its cell wall. These blebs functioned to facilitate material exchange with ectosymbiotic proto-mitochondria. The cytoplasm was then formed through the expansion of blebs around proto-mitochondria, with continuous spaces between the blebs giving rise to the endoplasmic reticulum, which later evolved into the eukaryotic secretory system. Further bleb-fusion steps yielded a continuous plasma membrane, which served to isolate the endoplasmic reticulum from the environment. The inside-out theory is consistent with diverse kinds of data and provides an alternative framework by which to explore and understand the dynamic organization of modern eukaryotic cells. It also helps to explain a number of previously enigmatic features of cell biology, including the autonomy of nuclei in syncytia and the subcellular localization of protein N-glycosylation, and makes many predictions, including a novel mechanism of interphase nuclear pore insertion.

  14. Predation and eukaryote cell origins: a coevolutionary perspective.

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    Cavalier-Smith, T

    2009-02-01

    Cells are of only two kinds: bacteria, with DNA segregated by surface membrane motors, dating back approximately 3.5Gy; and eukaryotes, which evolved from bacteria, possibly as recently as 800-850My ago. The last common ancestor of eukaryotes was a sexual phagotrophic protozoan with mitochondria, one or two centrioles and cilia. Conversion of bacteria (=prokaryotes) into a eukaryote involved approximately 60 major innovations. Numerous contradictory ideas about eukaryogenesis fail to explain fundamental features of eukaryotic cell biology or conflict with phylogeny. Data are best explained by the intracellular coevolutionary theory, with three basic tenets: (1) the eukaryotic cytoskeleton and endomembrane system originated through cooperatively enabling the evolution of phagotrophy; (2) phagocytosis internalised DNA-membrane attachments, unavoidably disrupting bacterial division; recovery entailed the evolution of the nucleus and mitotic cycle; (3) the symbiogenetic origin of mitochondria immediately followed the perfection of phagotrophy and intracellular digestion, contributing greater energy efficiency and group II introns as precursors of spliceosomal introns. Eukaryotes plus their archaebacterial sisters form the clade neomura, which evolved from a radically modified derivative of an actinobacterial posibacterium that had replaced the ancestral eubacterial murein peptidoglycan by N-linked glycoproteins, radically modified its DNA-handling enzymes, and evolved cotranslational protein secretion, but not the isoprenoid-ether lipids of archaebacteria. I focus on this phylogenetic background and on explaining how in response to novel phagotrophic selective pressures and ensuing genome internalisation this prekaryote evolved efficient digestion of prey proteins by retrotranslocation and 26S proteasomes, then internal digestion by phagocytosis, lysosomes, and peroxisomes, and eukaryotic vesicle trafficking and intracellular compartmentation.

  15. Effects of Nonequilibrium Plasmas on Eukaryotic Cells

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    2009-05-01

    with cellulosic plates under their cell membrane, the diatom C. hystrix has a siliceous cell wall and organic layer, while A. sanguinea is an...unarmored (naked) dinoflagellate, i.e. without the cellulosic plates (see SEM micrographs below). Some non-motile cells recovered their motility and...formed a somewhat reticulate or porous structure as a cell covering. In the A. sanguinea sample where pH was adjusted to 3.0 (corresponds to the pH

  16. Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

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    Dennig, Jörg

    Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

  17. Chromatin—a global buffer for eukaryotic gene control

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    Yuri M. Moshkin

    2015-09-01

    Full Text Available Most of eukaryotic DNA is embedded into nucleosome arrays formed by DNA wrapped around a core histone octamer. Nucleosome is a fundamental repeating unit of chromatin guarding access to the genetic information. Here, I will discuss two facets of nucleosome in eukaryotic gene control. On the one hand, nucleosome acts as a regulatory unit, which controls gene switches through a set of post-translational modifications occurring on histone tails. On the other hand, global configuration of nucleosome arrays with respect to nucleosome positioning, spacing and turnover acts as a tuning parameter for all genomic functions. A “histone code” hypothesis extents the Jacob-Monod model for eukaryotic gene control; however, when considering factors capable of reconfiguring entire nucleosome array, such as ATP-dependent chromatin remodelers, this model becomes limited. Global changes in nucleosome arrays will be sensed by every gene, yet the transcriptional responses might be specific and appear as gene targeted events. What determines such specificity is unclear, but it’s likely to depend on initial gene settings, such as availability of transcription factors, and on configuration of new nucleosome array state.

  18. Recognition of extremophilic archaeal viruses by eukaryotic cells

    DEFF Research Database (Denmark)

    Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur

    2016-01-01

    followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path......Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration...

  19. "Race for the Surface": Eukaryotic Cells Can Win.

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    Pham, Vy T H; Truong, Vi Khanh; Orlowska, Anna; Ghanaati, Shahram; Barbeck, Mike; Booms, Patrick; Fulcher, Alex J; Bhadra, Chris M; Buividas, Ričardas; Baulin, Vladimir; Kirkpatrick, C James; Doran, Pauline; Mainwaring, David E; Juodkazis, Saulius; Crawford, Russell J; Ivanova, Elena P

    2016-08-31

    With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants.

  20. Interaction of Low Temperature Plasmas with Prokaryotic and Eukaryotic Cells

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    Laroussi, Mounir

    2008-10-01

    Due to promising possibilities for their use in medical applications such as wound healing, surface modification of biocompatible materials, and the sterilization of reusable heat-sensitive medical instruments, low temperature plasmas and plasma jets are making big strides as a technology that can potentially be used in medicine^1-2. At this stage of research, fundamental questions about the effects of plasma on prokaryotic and eukaryotic cells are still not completely answered. An in-depth understanding of the pathway whereby cold plasma interact with biological cells is necessary before real applications can emerge. In this paper, first an overview of non-equilibrium plasma sources (both low and high pressures) will be presented. Secondly, the effects of plasma on bacterial cells will be discussed. Here, the roles of the various plasma agents in the inactivation process will be outlined. In particular, the effects of UV and that of various reactive species (O3, O, OH) are highlighted. Thirdly, preliminary findings on the effects of plasma on few types of eukaryotic cells will be presented. How plasma affects eukaryotic cells, such as mammalian cells, is very important in applications where the viability/preservation of the cells could be an issue (such as in wound treatment). Another interesting aspect is the triggering of apoptosis (programmed cell death). Some investigators have claimed that plasma is able to induce apoptosis in some types of cancer cells. If successfully replicated, this can open up a novel method of cancer treatment. In this talk however, I will briefly focus more on the wound healing potential of cold plasmas. ^1E. A. Blakely, K. A. Bjornstad, J. E. Galvin, O. R. Monteiro, and I. G. Brown, ``Selective Neuron Growth on Ion Implanted and Plasma Deposited Surfaces'', In Proc. IEEE Int. Conf. Plasma Sci., (2002), p. 253. ^2M. Laroussi, ``Non-thermal Decontamination of Biological Media by Atmospheric Pressure Plasmas: Review, Analysis, and

  1. Novel eukaryotic enzymes modifying cell-surface biopolymers

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    Aravind L

    2010-01-01

    Full Text Available Abstract Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA. We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1 the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58, which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber

  2. Novel eukaryotic enzymes modifying cell-surface biopolymers

    Science.gov (United States)

    2010-01-01

    Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA). We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p) that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1) the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58), which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber PMID:20056006

  3. The Evolution of Organellar Coat Complexes and Organization of the Eukaryotic Cell.

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    Rout, Michael P; Field, Mark C

    2017-06-20

    Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with β-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.

  4. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

    DEFF Research Database (Denmark)

    Nielsen, Olaf; Løbner-Olesen, Anders

    2008-01-01

    DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells...... prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism...... by using the model organisms Escherichia coli and Schizosaccharomyces pombe as examples. Although the underlying molecular details are different, the logic behind the control mechanisms is similar. For example, after initiation, crucial molecules required for the loading of replicative helicases in both...

  5. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Nielsen, Olaf; Løbner-Olesen, Anders

    2008-02-01

    DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells by using the model organisms Escherichia coli and Schizosaccharomyces pombe as examples. Although the underlying molecular details are different, the logic behind the control mechanisms is similar. For example, after initiation, crucial molecules required for the loading of replicative helicases in both prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism that prevents re-replication in both systems also increases the synthesis of DNA building blocks.

  6. Biological Influence of Deuterium on Procariotic and Eukaryotic Cells

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    Oleg Mosin

    2014-03-01

    Full Text Available Biologic influence of deuterium (D on cells of various taxonomic groups of prokaryotic and eukaryotic microorganisms realizing methylotrophic, chemoheterotrophic, photo-organotrophic, and photosynthetic ways of assimilation of carbon substrates are investigated at growth on media with heavy water (D2О. The method of step by step adaptation technique of cells to D2О was developed, consisting in plating of cells on 2 % agarose nutrient media containing increasing gradient of concentration of D2О (from 0 up to 98 % D2O and the subsequent selection of stable to D2O cells. In the result of that technique were obtained adapted to maximum concentration of D2O cells, biological material of which instead of hydrogen contained deuterium with levels of enrichment 92–97,5 at.% D.

  7. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

    OpenAIRE

    Nielsen, Olaf; Løbner-Olesen, Anders

    2008-01-01

    Udgivelsesdato: 2008-Feb DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells by using the model organisms Escherichia coli and Schizosaccharomyces pombe as examples. Although the underlying molecular details are different, the logic behind the con...

  8. THE COMPLEX ORGANIZATION OF EUKARYOTIC CELL NUCLEUS: THE NUCLEAR BODIES (I

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    Cristian Campeanu

    2012-10-01

    Full Text Available Identified short time after the discovery of cells, over 300 years ago, the cell nucleus of eukaryotes continuously focused the interest of scientists, which used increasingly sophisticated research tools to clarify its complex structure and functions. The results of all these studies, especially those carried out in the second half of the past century, proved and confirmed that the eukaryotic cell nucleus is the control center of all cellular activities and also ensures the continuity of genetic information along successive generations of cells. These vital functions are the result of selective expression of genes contained in the nuclear chromatin, which is a high ordered and dynamic structure, in permanent and bilateral relations with other nuclear components. Based on these considerations, the present review aims to synthetize the latest researches and concepts about the cell nuclear territory in three distinctive parts, according to the complexity of the topic

  9. An Interactive Exercise To Learn Eukaryotic Cell Structure and Organelle Function.

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    Klionsky, Daniel J.; Tomashek, John J.

    1999-01-01

    Describes a cooperative, interactive problem-solving exercise for studying eukaryotic cell structure and function. Highlights the dynamic aspects of movement through the cell. Contains 15 references. (WRM)

  10. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum.

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    Chen, Zhi-hui; Schaap, Pauline

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo.

  11. Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells.

    Science.gov (United States)

    Cox, Christopher R; Coburn, Phillip S; Gilmore, Michael S

    2005-02-01

    The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.

  12. Asymmetric cell division in polyploid giant cancer cells and low eukaryotic cells.

    Science.gov (United States)

    Zhang, Dan; Wang, Yijia; Zhang, Shiwu

    2014-01-01

    Asymmetric cell division is critical for generating cell diversity in low eukaryotic organisms. We previously have reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride demonstrate the ability to use an evolutionarily conserved process for renewal and fast reproduction, which is normally confined to simpler organisms. The budding yeast, Saccharomyces cerevisiae, which reproduces by asymmetric cell division, has long been a model for asymmetric cell division studies. PGCCs produce daughter cells asymmetrically in a manner similar to yeast, in that both use budding for cell polarization and cytokinesis. Here, we review the results of recent studies and discuss the similarities in the budding process between yeast and PGCCs.

  13. Bacterial scaffolds assemble novel higher-order complexes to reengineer eukaryotic cell processes.

    Science.gov (United States)

    Lesser, Cammie F; Leong, John M

    2011-07-05

    Many microbial pathogens use specialized secretion systems to inject proteins referred to as effectors directly into eukaryotic host cells. These effectors directly target various eukaryotic signaling pathways and cellular processes, often by mimicking the activity of host cell proteins. Effectors of pathogenic Escherichia coli and Salmonella typhimurium can also act as molecular scaffolds that not only recruit but also directly regulate the activity and localization of multiple eukaryotic proteins. By assembling and localizing disparate signaling pathways, the bacteria can reengineer host cell processes to generate novel processes not previously observed in eukaryotic cells.

  14. Eukaryotic checkpoints are absent in the cell division cycle of Entamoeba histolytica

    Indian Academy of Sciences (India)

    Sulagna Banerjee; Suchismita Das; Anuradha Lohia

    2002-11-01

    Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite, Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered in E. histolytica.

  15. Pi sensing and signalling: from prokaryotic to eukaryotic cells.

    Science.gov (United States)

    Qi, Wanjun; Baldwin, Stephen A; Muench, Stephen P; Baker, Alison

    2016-06-15

    Phosphorus is one of the most important macronutrients and is indispensable for all organisms as a critical structural component as well as participating in intracellular signalling and energy metabolism. Sensing and signalling of phosphate (Pi) has been extensively studied and is well understood in single-cellular organisms like bacteria (Escherichia coli) and Saccharomyces cerevisiae In comparison, the mechanism of Pi regulation in plants is less well understood despite recent advances in this area. In most soils the available Pi limits crop yield, therefore a clearer understanding of the molecular basis underlying Pi sensing and signalling is of great importance for the development of plants with improved Pi use efficiency. This mini-review compares some of the main Pi regulation pathways in prokaryotic and eukaryotic cells and identifies similarities and differences among different organisms, as well as providing some insight into future research.

  16. Structural and biomechanical basis of mitochondrial movement in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Wu M

    2013-10-01

    Full Text Available Min Wu,1 Aruna Kalyanasundaram,2 Jie Zhu1 1Laboratory of Biomechanics and Engineering, Institute of Biophysics, College of Science, Northwest A&F University, Yangling, Shaanxi, People's Republic of China; 2College of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA Abstract: Mitochondria serve as energy-producing organelles in eukaryotic cells. In addition to providing the energy supply for cells, the mitochondria are also involved in other processes, such as proliferation, differentiation, information transfer, and apoptosis, and play an important role in regulation of cell growth and the cell cycle. In order to achieve these functions, the mitochondria need to move to the corresponding location. Therefore, mitochondrial movement has a crucial role in normal physiologic activity, and any mitochondrial movement disorder will cause irreparable damage to the organism. For example, recent studies have shown that abnormal movement of the mitochondria is likely to be the reason for Charcot–Marie–Tooth disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, and schizophrenia. So, in the cell, especially in the particular polarized cell, the appropriate distribution of mitochondria is crucial to the function and survival of the cell. Mitochondrial movement is mainly associated with the cytoskeleton and related proteins. However, those components play different roles according to cell type. In this paper, we summarize the structural basis of mitochondrial movement, including microtubules, actin filaments, motor proteins, and adaptin, and review studies of the biomechanical mechanisms of mitochondrial movement in different types of cells. Keywords: mitochondrial movement, microtubules, actin filaments, motor proteins, adaptin

  17. The emerging roles of inositol pyrophosphates in eukaryotic cell physiology

    Indian Academy of Sciences (India)

    Swarna Gowri Thota; Rashna Bhandari

    2015-09-01

    Inositol pyrophosphates are water soluble derivatives of inositol that contain pyrophosphate or diphosphate moieties in addition to monophosphates. The best characterised inositol pyrophosphates, are IP7 (diphosphoinositol pentakisphosphate or PP-IP5), and IP8 (bisdiphosphoinositol tetrakisphosphate or (PP)2-IP4). These energy-rich small molecules are present in all eukaryotic cells, from yeast to mammals, and are involved in a wide range of cellular functions including apoptosis, vesicle trafficking, DNA repair, osmoregulation, phosphate homeostasis, insulin sensitivity, immune signalling, cell cycle regulation, and ribosome synthesis. Identified more than 20 years ago, there is still only a rudimentary understanding of the mechanisms by which inositol pyrophosphates participate in these myriad pathways governing cell physiology and homeostasis. The unique stereochemical and bioenergetic properties these molecules possess as a consequence of the presence of one or two pyrophosphate moieties in the vicinity of densely packed monophosphates are likely to form the molecular basis for their participation in multiple signalling and metabolic pathways. The aim of this review is to provide first time researchers in this area with an introduction to inositol pyrophosphates and a comprehensive overview on their cellular functions.

  18. Construction and identification of eukaryotic eukaryotic expression plasmid pcdna3.1-bace and its transient expression in cells

    Institute of Scientific and Technical Information of China (English)

    Huilin Gong; Guanjun Zhang; Weijiang Dong

    2006-01-01

    Objective: To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods: A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy-mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results: The results showed that the cDNAfragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully,and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion: The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the reason of Alzheimer's disease and lays the important foundation for preventing the AD laterly.

  19. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    NARCIS (Netherlands)

    Ortega, Alvaro D.; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles i

  20. Endocytosis and signaling: cell logistics shape the eukaryotic cell plan

    National Research Council Canada - National Science Library

    Sigismund, Sara; Confalonieri, Stefano; Ciliberto, Andrea; Polo, Simona; Scita, Giorgio; Di Fiore, Pier Paolo

    2012-01-01

    ... with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved...

  1. Inefficient complement system clearance of Trypanosoma cruzi metacyclic trypomastigotes enables resistant strains to invade eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Igor Cestari

    Full Text Available The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1 which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2 the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3 whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.

  2. Soil fertility controls the size-specific distribution of eukaryotes.

    Science.gov (United States)

    Mulder, Christian

    2010-05-01

    The large range of body-mass values of soil organisms provides a tool to assess the organization of soil ecological communities. Relationships between log-transformed body mass M and log-transformed numerical abundance N of all eukaryotes occurring under organic pastures, mature grasslands, and seminatural heathlands in the Netherlands were investigated. The observed allometry of (M,N) assemblages of below-ground communities strongly reflects the availability of primary macronutrients and essential micronutrients. This log-linear model describes the continuous variation in the allometric slope of animals and fungi along an increasing soil fertility gradient. The aggregate contribution of small invertebrates (M soil explains 72% of these shifts but the nitrogen concentration explains only 36%, with copper and zinc as intermediate predictors (59% and 49%, respectively). Empirical evidence supports common responses of invertebrates to the rates of resource supply and, possibly, to the above-ground primary production of ecosystems.

  3. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

    2015-04-30

    We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.

  4. Peptides encoded by short ORFs control development and define a new eukaryotic gene family.

    Directory of Open Access Journals (Sweden)

    Máximo Ibo Galindo

    2007-05-01

    Full Text Available Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal, a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs, which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.

  5. Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    SHANG Dan; ZHENG Qichang; SONG Zifang; LI Yiqing; WANG Xiedan; GUO Xingjun

    2006-01-01

    The eukaryotic expression of human arresten geneand its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells,while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-αactin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P<0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

  6. [Expression of target gene in eukaryotic cells driven by prokaryotic T7 promoter and its RNA polymerase].

    Science.gov (United States)

    Yuan, Zhi-Gang; Zhang, Jin-Ping; Chu, Yi-Wei; Wang, Ying; Xu, Wei; Xiong, Si-Dong

    2005-03-01

    To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.

  7. Peroxicretion: a novel secretion pathway in the eukaryotic cell

    Directory of Open Access Journals (Sweden)

    Luesken Francisca A

    2009-05-01

    Full Text Available Abstract Background Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag. The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

  8. Controlling segregation speed of entangled polymers by the shapes: A simple model for eukaryotic chromosome segregation

    Science.gov (United States)

    Sakai, Yuji; Tachikawa, Masashi; Mochizuki, Atsushi

    2016-10-01

    We report molecular dynamics simulations of the segregation of two overlapping polymers motivated by chromosome segregation in biological cells. We investigate the relationship between polymer shapes and segregation dynamics and show that elongation and compaction make entangled polymers segregate rapidly. This result suggests that eukaryotic chromosomes take such a characteristic rod-shaped structure, which is induced by condensins, to achieve rapid segregation.

  9. Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

    Science.gov (United States)

    Garg, Sriram G; Martin, William F

    2016-07-02

    Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically

  10. Recognition of extremophilic archaeal viruses by eukaryotic cells

    DEFF Research Database (Denmark)

    Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur;

    2016-01-01

    followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path...

  11. A 3D Hydrodynamic Model for Cytokinesis of Eukaryotic Cells

    Science.gov (United States)

    2014-08-01

    during cytokinesis, surface tension of the cell membrane also contributes to this process by retaining the morphological integrity of the offspring...during cytokinesis, surface tension of the cell membrane also contributes to this process by retaining the morphological integrity of the offspring...transformations and eventually divides into two or more offspring cells. For prokaryotic cells, the cell proliferation process is called binary

  12. Free energy difference in indolicidin attraction to eukaryotic and prokaryotic model cell membranes.

    Science.gov (United States)

    Yeh, In-Chul; Ripoll, Daniel R; Wallqvist, Anders

    2012-03-15

    We analyzed the thermodynamic and structural determinants of indolicidin interactions with eukaryotic and prokaryotic cell membranes using a series of atomistically detailed molecular dynamics simulations. We used quartz-supported bilayers with two different compositions of zwitterionic and anionic phospholipids as model eukaryotic and prokaryotic cell membranes. Indolicidin was preferentially attracted to the model prokaryotic cell membrane in contrast to the weak adsorption on the eukaryotic membrane. The nature of the indolicidin surface adsorption depended on an electrostatic guiding component, an attractive enthalpic component derived from van der Waals interactions, and a balance between entropic factors related to peptide confinement at the interface and counterion release from the bilayer surface. Thus, whereas we attributed the specificity of the indolicidin/membrane interaction to electrostatics, these interactions were not the sole contributors to the free energy of adsorption. Instead, a balance between an attractive van der Waals enthalpic component and a repulsive entropic component determined the overall strength of indolicidin adsorption.

  13. Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

    NARCIS (Netherlands)

    Egorova-Zachernyuk, T.A.; Bosman, G.J.C.G.M.; Pistorius, A.M.A.; Grip, W.J. de

    2009-01-01

    Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we e

  14. Evolutionarily conserved binding of translationally controlled tumor protein to eukaryotic elongation factor 1B.

    Science.gov (United States)

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-04-03

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Membrane tension feedback on shape and motility of eukaryotic cells

    Science.gov (United States)

    Winkler, Benjamin; Aranson, Igor S.; Ziebert, Falko

    2016-04-01

    In the framework of a phase field model of a single cell crawling on a substrate, we investigate how the properties of the cell membrane affect the shape and motility of the cell. Since the membrane influences the cell dynamics on multiple levels and provides a nontrivial feedback, we consider the following fundamental interactions: (i) the reduction of the actin polymerization rate by membrane tension; (ii) area conservation of the cell's two-dimensional cross-section vs. conservation of the circumference (i.e. membrane inextensibility); and (iii) the contribution from the membrane's bending energy to the shape and integrity of the cell. As in experiments, we investigate two pertinent observables - the cell's velocity and its aspect ratio. We find that the most important effect is the feedback of membrane tension on the actin polymerization. Bending rigidity has only minor effects, visible mostly in dynamic reshaping events, as exemplified by collisions of the cell with an obstacle.

  16. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

    Science.gov (United States)

    Yu, Changming; Pike, Gennett M; Rinkoski, Tommy A; Correia, Cristina; Kaufmann, Scott H; Federspiel, Mark J

    2015-08-11

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

  17. Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking.

    Science.gov (United States)

    Hopper, Anita K; Huang, Hsiao-Yun

    2015-06-01

    tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Cationic amphiphiles as delivery system for genes into eukaryotic cells

    NARCIS (Netherlands)

    Oberle, Volker; Zuhorn, Inge S.; Audouy, Sandrine; Bakowsky, Udo; Smisterová, Jarmila; Engberts, Jan B.F.N.; Hoekstra, Dick; Gregoriadis, G; McCormack, B

    2000-01-01

    Cationic liposomes, consisting of synthetic amphiphiles and a so-called helper lipid, rapidly form complexes with DNA, known as lipoplexes. When incubated with cells in culture, the DNA can be delivered into the cell and becomes expressed. Because of these properties, lipoplexes are considered a use

  19. New technique for needle-less implantation of eukaryotic cells

    NARCIS (Netherlands)

    Silva, L.F. Arenas da; Schober, L.; Sloff, M.; Traube, A.; Hart, M.L.; Feitz, W.F.J.; Stenzl, A.

    2015-01-01

    BACKGROUND AIMS: On review of the use of stem cells in the literature, promissory outcomes for functional organ recovery in many subspecialties in medicine underscore its therapeutic potential. The application of stem cells through the use of a needle can result in additional scar formation, which i

  20. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

    DEFF Research Database (Denmark)

    Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris;

    2016-01-01

    for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA...

  1. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

    DEFF Research Database (Denmark)

    Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris

    2016-01-01

    for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA...... circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types....

  2. Peroxicretion: a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, C.M.J.; Ten Haaft, P.J.T; Minneboo, I.M.; Hartog, M.P.; Damveld, R.A.; Van der Laan, J.M.; Akeroyd, M; Wenzel, T.J.; Luesken, F.A.; Veenhuis, M.; Van der Klei, I.; De Winde, J.H.

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  3. Peroxicretion : a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, Cees M.J.; Haaft, Peter J. ten; Minneboo, Ingeborg M.; Hartog, Miranda P.; Damveld, Robbert A.; Laan, Jan Metske van der; Akeroyd, Michiel; Wenzel, Thibaut J.; Luesken, Francisca A.; Veenhuis, Marten; Klei, Ida van der; Winde, Johannes H. de

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  4. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    Science.gov (United States)

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  5. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells.

    Science.gov (United States)

    Møller, Henrik D; Bojsen, Rasmus K; Tachibana, Chris; Parsons, Lance; Botstein, David; Regenberg, Birgitte

    2016-04-04

    Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

  6. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Directory of Open Access Journals (Sweden)

    Andreas K Brödel

    Full Text Available Internal ribosome entry site (IRES elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR of the Cricket paralysis virus (CrPV genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established

  7. HPMA and HEMA copolymer bead interactions with eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Cristina D. Vianna-Soares

    2004-09-01

    Full Text Available Two different hydrophilic acrylate beads were prepared via aqueous suspension polymerization. Beads produced of a hydroxypropyl methacrylate (HPMA and ethyleneglycol methacrylate (EDMA copolymer were obtained using a polyvinyl alcohol suspending medium. Copolymers of 2hydroxyethyl methacrylate (HEMA, methyl methacrylate (MMA and ethyleneglycol methacrylate (EDMA beads were obtained using magnesium hydroxide as the suspending agent. Following characterization by scanning electron microscopy (SEM, nitrogen sorption analysis (NSA and mercury intrusion porosimetry (MIP, the beads were cultured with monkey fibroblasts (COS7 to evaluate their ability to support cell growth, attachment and adhesion. Cell growth behavior onto small HPMA/EDMA copolymer beads and large HEMA/MMA/EDMA copolymer beads is evaluated regarding their hidrophilicity/hidrophobicity and surface roughness.

  8. Systems-biology dissection of eukaryotic cell growth

    Directory of Open Access Journals (Sweden)

    Andrews Justen

    2010-05-01

    Full Text Available Abstract A recent article in BMC Biology illustrates the use of a systems-biology approach to integrate data across the transcriptome, proteome and metabolome of budding yeast in order to dissect the relationship between nutrient conditions and cell growth. See research article http://jbiol.com/content/6/2/4 and http://www.biomedcentral.com/1741-7007/8/68

  9. Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

    Directory of Open Access Journals (Sweden)

    Jing Dan

    2017-03-01

    Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

  10. Surface glycosaminoglycans protect eukaryotic cells against membrane-driven peptide bacteriocins.

    Science.gov (United States)

    Martín, Rebeca; Escobedo, Susana; Martín, Carla; Crespo, Ainara; Quiros, Luis M; Suarez, Juan E

    2015-01-01

    Enzymatic elimination of surface glycosaminoglycans or inhibition of their sulfation provokes sensitizing of HT-29 and HeLa cells toward the peptide bacteriocins nisin A, plantaricin C, and pediocin PA-1/AcH. The effect can be partially reversed by heparin, which also lowers the susceptibility of Lactococcus lactis to nisin A. These data indicate that the negative charge of the glycosaminoglycan sulfate residues binds the positively charged bacteriocins, thus protecting eukaryotic cells from plasma membrane damage.

  11. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  12. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  13. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells.

    Science.gov (United States)

    Ortega, Alvaro D; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  14. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Álvaro D. Ortega

    2014-11-01

    Full Text Available Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of ‘intact’ intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  15. Ion Channels Activated by Mechanical Forces in Bacterial and Eukaryotic Cells.

    Science.gov (United States)

    Sokabe, Masahiro; Sawada, Yasuyuki; Kobayashi, Takeshi

    2015-01-01

    Since the first discovery of mechanosensitive ion channel (MSC) in non-sensory cells in 1984, a variety of MSCs has been identified both in prokaryotic and eukaryotic cells. One of the central issues concerning MSCs is to understand the molecular and biophysical mechanisms of how mechanical forces activate/open MSCs. It has been well established that prokaryotic (mostly bacterial) MSCs are activated exclusively by membrane tension. Thus the problem to be solved with prokaryotic MSCs is the mechanisms how the MSC proteins receive tensile forces from the lipid bilayer and utilize them for channel opening. On the other hand, the activation of many eukaryotic MSCs crucially depends on tension in the actin cytoskeleton. By using the actin cytoskeleton as a force sensing antenna, eukaryotic MSCs have obtained sophisticated functions such as remote force sensing and force-direction sensing, which bacterial MSCs do not have. Actin cytoskeletons also give eukaryotic MSCs an interesting and important function called "active touch sensing", by which cells can sense rigidity of their substrates. The contractile actin cytoskeleton stress fiber (SF) anchors its each end to a focal adhesion (FA) and pulls the substrate to generate substrate-rigidity-dependent stresses in the FA. It has been found that those stresses are sensed by some Ca2+-permeable MSCs existing in the vicinity of FAs, thus the MSCs work as a substrate rigidity sensor that can transduce the rigidity into intracellular Ca2+ levels. This short review, roughly constituting of two parts, deals with molecular and biophysical mechanisms underlying the MSC activation process mostly based on our recent studies; (1) structure-function in bacterial MSCs activation at the atomic level, and (2) roles of actin cytoskeletons in the activation of eukaryotic MSCs.

  16. Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

    Science.gov (United States)

    Schauer, Grant D.; O’Donnell, Michael E.

    2017-01-01

    The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand. PMID:28069954

  17. Regulation of eukaryotic initiation factor 4AII by MyoD during murine myogenic cell differentiation.

    Directory of Open Access Journals (Sweden)

    Gabriela Galicia-Vázquez

    Full Text Available Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

  18. An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

    Science.gov (United States)

    Centrella, Michael; Porter, David L; McCarthy, Thomas L

    2011-08-15

    Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.

  19. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

    Science.gov (United States)

    Guo, Junjie U.; Bartel, David P.

    2017-01-01

    In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in post-transcriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex–forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex–forming sequences. PMID:27708011

  20. The scale-free dynamics of eukaryotic cells.

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    Miguel A Aon

    Full Text Available Temporal organization of biological processes requires massively parallel processing on a synchronized time-base. We analyzed time-series data obtained from the bioenergetic oscillatory outputs of Saccharomyces cerevisiae and isolated cardiomyocytes utilizing Relative Dispersional (RDA and Power Spectral (PSA analyses. These analyses revealed broad frequency distributions and evidence for long-term memory in the observed dynamics. Moreover RDA and PSA showed that the bioenergetic dynamics in both systems show fractal scaling over at least 3 orders of magnitude, and that this scaling obeys an inverse power law. Therefore we conclude that in S. cerevisiae and cardiomyocytes the dynamics are scale-free in vivo. Applying RDA and PSA to data generated from an in silico model of mitochondrial function indicated that in yeast and cardiomyocytes the underlying mechanisms regulating the scale-free behavior are similar. We validated this finding in vivo using single cells, and attenuating the activity of the mitochondrial inner membrane anion channel with 4-chlorodiazepam to show that the oscillation of NAD(PH and reactive oxygen species (ROS can be abated in these two evolutionarily distant species. Taken together these data strongly support our hypothesis that the generation of ROS, coupled to redox cycling, driven by cytoplasmic and mitochondrial processes, are at the core of the observed rhythmicity and scale-free dynamics. We argue that the operation of scale-free bioenergetic dynamics plays a fundamental role to integrate cellular function, while providing a framework for robust, yet flexible, responses to the environment.

  1. A comparison of autogenous theories for the origin of eukaryotic cells.

    Science.gov (United States)

    Baum, David A

    2015-12-01

    Eukaryotic cells have many unique features that all evolved on the stem lineage of living eukaryotes, making it difficult to reconstruct the order in which they accumulated. Nuclear endosymbiotic theories hold that three prokaryotes (nucleus, cytoplasm, and mitochondrion) came together to form a eukaryotic cell, whereas autogenous models hold that the nucleus and cytoplasm formed through evolutionary changes in a single prokaryotic lineage. Given several problems with nuclear endosymbiotic theories, this review focuses on autogenous models. Until recently all autogenous models assumed an outside-in (OI) topology, proposing that the nuclear envelope was formed from membrane-bound vesicles within the original cell body. Buzz Baum and I recently proposed an inside-out (IO) alternative, suggesting that the nucleus corresponds to the original cell body, with the cytoplasmic compartment deriving from extracellular protrusions. In this review, I show that OI and IO models are compatible with both mitochondria early (ME) or mitochondria late (ML) formulations. Whereas ME models allow that the relationship between mitochondria and host was mutualistic from the outset, ML models imply that the association began with predation or parasitism, becoming mutualistic later. In either case, the mutualistic interaction that eventually formed was probably syntrophic. Diverse features of eukaryotic cell biology align well with the IOME model, but it would be premature to rule out the OIME model. ML models require that phagocytosis, a complex and energy expensive process, evolved before mitochondria, which seems unlikely. Nonetheless, further research is needed, especially resolution of the phylogenetic affinities of mitochondria. © 2015 Botanical Society of America.

  2. The origin of the eukaryotic cell based on conservation of existing interfaces.

    Science.gov (United States)

    de Roos, Albert D G

    2006-01-01

    Current theories about the origin of the eukaryotic cell all assume that during evolution a prokaryotic cell acquired a nucleus. Here, it is shown that a scenario in which the nucleus acquired a plasma membrane is inherently less complex because existing interfaces remain intact during evolution. Using this scenario, the evolution to the first eukaryotic cell can be modeled in three steps, based on the self-assembly of cellular membranes by lipid-protein interactions. First, the inclusion of chromosomes in a nuclear membrane is mediated by interactions between laminar proteins and lipid vesicles. Second, the formation of a primitive endoplasmic reticulum, or exomembrane, is induced by the expression of intrinsic membrane proteins. Third, a plasma membrane is formed by fusion of exomembrane vesicles on the cytoskeletal protein scaffold. All three self-assembly processes occur both in vivo and in vitro. This new model provides a gradual Darwinistic evolutionary model of the origins of the eukaryotic cell and suggests an inherent ability of an ancestral, primitive genome to induce its own inclusion in a membrane.

  3. Novel electrochemical sensor system for monitoring metabolic activity during the growth and cultivation of prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Pescheck, M; Schrader, J; Sell, D

    2005-09-01

    A novel amperometric sensor system is presented which directly reflects the metabolic activity of prokaryotic and eukaryotic cells during cultivation. The principle of an externally mounted sensor is current measurement using a three-electrode system. Only living cells are detected since the current signal is based on a redox mediator. Added to a culture sample in its oxidized form, the mediator is reduced by cellular metabolism and subsequently re-oxidized at the anode. The spontaneous immobilisation of the cells in the reaction vessel of the sensor by swelling dextrane polymers (Sephadex) prior to measurement is the key to a fast, consistent signal. Even metabolically less active mammalian cells produce a reliable signal within a few minutes; this may open up future applications of the electrochemical sensor in closed loop process control not only for bacterial and fungal bioprocesses, but also in cell culture technology.

  4. Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

    DEFF Research Database (Denmark)

    Goldar, A.; Arneodo, A.; Audit, B.

    2016-01-01

    We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations......, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement...... with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic...

  5. Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

    Science.gov (United States)

    Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

    2016-03-01

    We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

  6. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Directory of Open Access Journals (Sweden)

    Michał Arabski

    2012-01-01

    Full Text Available Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

  7. THE COMPLEX ORGANIZATION OF EUKARYOTIC CELL NUCLEUS (III: THE NUCLEAR MATRIX AND THE NUCLEAR LAMINA

    Directory of Open Access Journals (Sweden)

    Cristian S. Cimpeanu

    2013-07-01

    Full Text Available A large variety of nuclear fibrous proteins (such as actin, myosin, lamin B, transcription factors, topoisomerases, etc represent constitutive elements of complex structures present in the eukaryotic nuclei: the nuclear matrix and the nuclear lamina, repectively. These nuclear compartments, with fibrous network-like structure, play crucialroles in structural organization of nuclei, chromatin remodeling, DNA transcription, signals transduction, cell cycle regulation, embryonic development and other nuclear basic processes.

  8. Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology.

    Science.gov (United States)

    Lemonnier, Marc; Landraud, Luce; Lemichez, Emmanuel

    2007-09-01

    Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.

  9. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

    2014-05-14

    Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts.

  10. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    Science.gov (United States)

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  11. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Carlos Medina

    Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

  12. Invasive cells in animals and plants: searching for LECA machineries in later eukaryotic life

    Science.gov (United States)

    2013-01-01

    Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants – two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA. Reviewer: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia. PMID:23557484

  13. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric

    2010-01-01

    lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ¿T6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions......Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial...... cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain...

  14. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  15. Cells under pressure - treatment of eukaryotic cells with high hydrostatic pressure, from physiologic aspects to pressure induced cell death.

    Science.gov (United States)

    Frey, Benjamin; Janko, Christina; Ebel, Nina; Meister, Silke; Schlücker, Eberhard; Meyer-Pittroff, Roland; Fietkau, Rainer; Herrmann, Martin; Gaipl, Udo S

    2008-01-01

    The research on high hydrostatic pressure in medicine and life sciences is multifaceted. According to the used pressure head the research has to be divided into two different parts. To study physiological aspects of pressure on eukaryotic cells physiological pressure (pHHP; highly reversible alterations and normally does not affect cellular viability. The treatment of eukaryotic cells with non-physiological pressure (HHP; > or = 100 MPa) reveals different outcomes. Treatment with HHP or = 200 MPa. Moreover, HHP treatment with > 300 MPa leads to necrosis. Therefore, HHP plays a role for the sterilisation of human transplants, of food stuff, and pharmaceuticals. Human tumour cells subjected to HHP > 300 MPa display a necrotic phenotype along with a gelificated cytoplasm, preserve their shape, and retain their immunogenicity. These observations favour the use of HHP to produce whole cell based tumour vaccines. Further experiments revealed that the increment of pressure as well as the pressure holding time influences the cell death of tumour cells. We conclude that high hydrostatic pressure offers both, an economic, easy to apply, clean, and fast technique for the generation of vaccines, and a promising tool to study physiological aspects.

  16. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

    Directory of Open Access Journals (Sweden)

    Sandra Schwarz

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  17. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    Directory of Open Access Journals (Sweden)

    Nicola Vitulo

    2007-01-01

    Full Text Available The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed.

  18. Optical propulsion of mammalian eukaryotic cells on an integrated channel waveguide

    Science.gov (United States)

    Shahimin, M. Mohamad; Perney, N. M. B.; Brooks, S.; Hanley, N.; Wright, K. L.; Wilkinson, J. S.; Melvin, T.

    2011-02-01

    The optical propulsion of mammalian eukaryotic cells along the surface of an integrated channel waveguide is demonstrated. 10μm diameter polymethylmethacrylate (PMMA) spherical particles and similarly sized mammalian eukaryotic cells in aqueous medium are deposited in a reservoir over a caesium ion-exchanged channel waveguide. Light from a fibre laser at 1064nm was coupled into the waveguide, causing the polymer particles or cells to be propelled along the waveguide at a velocity which is dependent upon the laser power. A theoretical model was used to predict the propulsion velocity as a function of the refractive index of the particle. The experimental results obtained for the PMMA particles and the mammalian cells show that for input powers greater than 50mW the propulsion velocity is approximately that obtained by the theoretical model. For input powers of less than ~50mW neither particles nor cells were propelled; this is considered to be a result of surface forces (which are not considered in the theoretical model). The results are discussed in light of the potential application of optical channel waveguides for bioanalytical applications, namely in the identification and sorting of mammalian cells from mixed populations without the need for fluorescence or antibody labels.

  19. Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris.

    Science.gov (United States)

    Matin, Abdul; Jeong, Seok Ryoul; Faull, Jane; Rivas, Antonio Ortega; Khan, Naveed Ahmed

    2006-10-01

    Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.

  20. Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.

    Science.gov (United States)

    Celedon, Jose M; Cline, Kenneth

    2013-02-01

    Protein trafficking and localization in plastids involve a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called 'conservative sorting'. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  1. Folded genome as a platform for the functional compartmentalization of the eukaryotic cell nucleus

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2014-03-01

    Full Text Available In a number of recent studies a tight interconnection between the spatial organization of the eukaryotic genome and its functioning has been demonstrated. Moreover, it is becoming evident that the folded DNA by itself consti- tutes an important, if not the key, factor supporting the internal nuclear organization. In this review, we will discuss the current state of chromatin research with the special attention focused on chromosome territories, chromatin folding and dynamics, chromatin domains, transcription and replication factories. Based on this analysis we will show how interphase chromosomes define the assembly of different nuclear compartments and underlie the spatial compartmentalization of the cell nucleus.

  2. Functional expression of human leukocyte elastase (HLE)/medullasin in eukaryotic cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Shimizu, H; Naruto, M

    1990-03-30

    We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.

  3. Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections

    Directory of Open Access Journals (Sweden)

    Rachid A. El-Aouar Filho

    2017-05-01

    Full Text Available Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

  4. The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells.

    Science.gov (United States)

    Coburn, Phillip S; Gilmore, Michael S

    2003-10-01

    The enterococcal cytolysin, a two-peptide lytic system, is a divergent relative of a large family of toxins and bacteriocins secreted by pathogenic and non-pathogenic Gram-positive bacteria. This family includes the lantibiotics and streptolysin S. The enterococcal cytolysin is of interest because its activities enhance enterococcal virulence in infection models and, in epidemiological studies, it has been associated with patient mortality. The cytolysin is lethal for a broad range of prokaryotic and eukaryotic cells, and this activity requires two non-identical, post-translationally modified peptides. The smaller of the two peptides also plays a role in a quorum-sensing autoinduction of the cytolysin operon. As a trait that is present in particularly virulent strains of Enterococcus faecalis, including strains that are resistant to multiple antibiotics, it serves as a model for testing the value of developing new virulence-targeting therapeutics. Further, because of the interest in small membrane active peptides as therapeutics themselves, studies of the molecular structure/activity relationships for the cytolysin peptides are providing insights into the physical basis for prokaryotic versus eukaryotic cell targeting.

  5. Cellular differentiation in the process of generation of the eukaryotic cell

    Science.gov (United States)

    Nakamura, Hakobu; Hase, Atsushi

    1990-11-01

    Primitive atmosphere of the earth did not contain oxygen gas (O2) when the proto-cells were generated successfully as the resut of chemical evolution and then evolved. Therefore, they first had acquired anaerobic energy metabolism, fermentation. The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions. Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle. On the other hand, O2-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem. These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacteriumChlorogloea fritschii and aerobic photosynthetic bacteriaRhodospirillum rubrum andErythrobacter sp. Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium. Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O2 -respiratory enzymes show similarities to corresponding prokaryotic enzymes. However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules. In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only

  6. The Eukaryotic Cell Originated in the Integration and Redistribution of Hyperstructures from Communities of Prokaryotic Cells Based on Molecular Complementarity

    OpenAIRE

    Vic Norris; Robert Root-Bernstein

    2009-01-01

    In the “ecosystems-first” approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entaili...

  7. Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels.

    Directory of Open Access Journals (Sweden)

    Valérie Panneels

    Full Text Available BACKGROUND: Membrane proteins (MPs play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs. Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. PRINCIPAL FINDINGS: We describe the homologous and heterologous expression of 10 different targets from the three major MP classes--G protein-coupled receptors (GPCRs, transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2-0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter--both involved in synaptic transmission--showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. SIGNIFICANCE: We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in

  8. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  9. Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Ya-pei; XU Chun-xiao; HOU Guo-sheng; XIN Jia-xuan; WANG Wei; LIU Xian-xi

    2010-01-01

    Background Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein.The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.Methods The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN.Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting.Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry.To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.Results DNA sequence confirmed that pcDNA-tumstatin was successfully constructed.RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively.After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase.And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.Conclusions Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

  10. Applications of Recombinant Dna Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part B: Eukaryotic Gene Transcription and Post-Transcripional Rna Processing

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The transcription of DNA into RNA is the primary level at which gene expression is controlled in eukaryotic cells. Eukaryotic gene transcription  involves several different RNA polymerases that interact with a host of transcription factors to initiate transcription. Genes that encode proteins are transcribed into messenger RNA (mRNA by RNA polymerase II. Ribosomal RNAs (rRNAs and transfer RNAs (tRNAs are transcribed by RNA polymerase I and III, respectively.  The production of each mRNA in human cells involves complex interactions of proteins (ie, trans-acting factors with specific sequences on the DNA (ie, cis-acting elements. Cis-acting elements are short base sequences adjacent to or within a particular gene. While the regulation of transcription is a pivotal step in the control of gene expression, a variety of molecular events, collectively known as ’RNA processing’  add an additional level of control of gene expression in eukaryotic cells.

  11. Invasion of Eukaryotic Cells by Legionella Pneumophila: A Common Strategy for all Hosts?

    Directory of Open Access Journals (Sweden)

    Paul S Hoffman

    1997-01-01

    Full Text Available Legionella pneumophila is an environmental micro-organism capable of producing an acute lobar pneumonia, commonly referred to as Legionnaires’ disease, in susceptible humans. Legionellae are ubiquitous in aquatic environments, where they survive in biofilms or intracellularly in various protozoans. Susceptible humans become infected by breathing aerosols laden with the bacteria. The target cell for human infection is the alveolar macrophage, in which the bacteria abrogate phagolysosomal fusion. The remarkable ability of L pneumophila to infect a wide range of eukaryotic cells suggests a common strategy that exploits very fundamental cellular processes. The bacteria enter host cells via coiling phagocytosis and quickly subvert organelle trafficking events, leading to formation of a replicative phagosome in which the bacteria multiply. Vegetative growth continues for 8 to 10 h, after which the bacteria develop into a short, highly motile form called the ‘mature form’. The mature form exhibits a thickening of the cell wall, stains red with the Gimenez stain, and is between 10 and 100 times more infectious than agar-grown bacteria. Following host cell lysis, the released bacteria infect other host cells, in which the mature form differentiates into a Gimenez-negative vegetative form, and the cycle begins anew. Virulence of L pneumophila is considered to be multifactorial, and there is growing evidence for both stage specific and sequential gene expression. Thus, L pneumophila may be a good model system for dissecting events associated with the host-parasite interactions.

  12. Anisotropic diffusion of macromolecules in the contiguous nucleocytoplasmic fluid during eukaryotic cell division.

    Science.gov (United States)

    Pawar, Nisha; Donth, Claudia; Weiss, Matthias

    2014-08-18

    Character and rapidity of protein diffusion in intracellular fluids are key determinants of the dynamics and steady state of a plethora of biochemical reactions. So far, an anomalous diffusion in cytoplasmic fluids with viscoelastic and even glassy characteristics has been reported in a variety of organisms on several length scales and timescales. Here, we show that the contiguous fluid of former cytoplasm and nucleoplasm features an anisotropically varying diffusion of macromolecules during eukaryotic cell division. In metaphase, diffusion in the contiguous nucleocytoplasmic fluid appears less anomalous along the spindle axis as compared to perpendicular directions. As a consequence, the long-time diffusion of macromolecules preferentially points along the spindle axis, leading to prolonged residence of macromolecules in the spindle region. Based on our experimental data, we suggest that anisotropic diffusion facilitates the encounter and interaction of spindle-associated proteins, e.g., during the formation of a dynamic spindle matrix. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Cytotoxic and apoptotic effects of scorpion Leiurus quinquestriatus venom on 293T and C2C12 eukaryotic cell lines

    Directory of Open Access Journals (Sweden)

    M. A. A. Omran

    2003-01-01

    Full Text Available Scorpion venom toxicity is of major concern due to its influence on human activities and public health. The cytotoxicity and apoptosis induced by scorpion L. quinquestriatus venom on two established eukaryotic cell lines (293T and C2C12 were analyzed. Both cultured cell lines were incubated with varying doses (10, 20, and 50 µg/ml of scorpion venom in serum free medium (SFM for 0.5, 1, 2, 4, and 8 hours at 37°C. The percentage of total lactate dehydrogenase (LDH released in the culture during venom incubation was used as an index of cell damage. Control culture was treated with an equal amount of SFM. Cell injury was recognized morphologically and apoptosis was researched by a Fluorescing Apoptosis Detection System using the principle of TUNEL (TdT-mediated dUTP Nick-End Labelling assay and confirmed by another assay concerning nuclear DNA staining with DAPI stain. Cytotoxicity was remarkable and cell survival highly reduced at the highest tested concentration (50 µg/ml. These effects were rapid and observed within 30 minutes. The apparent initial damage to the nucleus and lysis of the plasmalemma and/or organelle membranes, which was evident by a significant increase in cytosolic LDH release, suggested that this toxin acts at the membrane level. The morphological changes that occurred in apoptotic cells include condensation and compartmentalization of nuclear and cytoplasmic materials into structurally preserved membrane-bound fragments or blebs. The cytotoxic effects are dose and time dependent and cell death by apoptosis was more characteristic of 293T cells than C2C12 cells. The apoptotic effects were more prominent and clear in the early stages of toxicity, while other forms of cell damage such as swelling, rupture, and/or necrosis occurred at later stages.

  14. UCP2- and non-UCP2-mediated electric current in eukaryotic cells exhibits different properties.

    Science.gov (United States)

    Wang, Ruihua; MoYung, K C; Zhang, M H; Poon, Karen

    2015-12-01

    Using live eukaryotic cells, including cancer cells, MCF-7 and HCT-116, normal hepatocytes and red blood cells in anode and potassium ferricyanide in cathode of MFC could generate bio-based electric current. Electrons and protons generated from the metabolic reaction in both cytosol and mitochondria contributing to the leaking would mediate the generation of electric current. Both resveratrol (RVT) and 2,4-dinitrophenol (DNP) used to induce proton leak in mitochondria were found to promote electric current production in all cells except red blood cells without mitochondria. Proton leak might be important for electric current production by bringing the charge balance in cells to enhance the further electron leak. The induced electric current by RVT can be blocked by Genipin, an inhibitor of UCP2-mediated proton leak, while that induced by DNP cannot. RVT could reduce reactive oxygen species (ROS) level in cells better than that of DNP. In addition, RVT increased mitochondrial membrane potential (MMP), while DNP decreased it. Results highly suggested the existence of at least two types of electric current that showed different properties. They included UCP2-mediated and non-UCP2-mediated electric current. UCP2-mediated electric current exhibited higher reactive oxygen species (ROS) reduction effect per unit electric current production than that of non-UCP2-mediated electric current. Higher UCP2-mediated electric current observed in cancer cells might contribute to the mechanism of drug resistence. Correlation could not be established between electric current production with either ROS and MMP without distinguishing the types of electric current.

  15. Bacterial Toxin Modulation of the Eukaryotic Cell Cycle: Are all Cytolethal Distending Toxins Created Equally?

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    Amandeep eGargi

    2012-10-01

    Full Text Available The cytolethal distending toxins (CDTs comprise a family of intracellular-acting bacterial protein toxins whose actions upon eukaryotic cells result in several consequences, the most characteristic of which is the induction of G2/M cell cycle arrest. Most CDTs are hetero-tripartite assemblies of CdtA, CdtB, and CdtC, with CdtB required for CDT-mediated cell cycle arrest. Several lines of evidence indicate that CdtA and CdtC are required for the optimal intracellular activity of CdtB, although the exact functional roles of CdtA and CdtC remain poorly understood. The genes encoding the CDTs have been identified in a diverse array of Gram-negative pathogenic bacteria. More recently, the genes encoding several CdtB subunits have been associated with alternatively linked subunits resembling the B-subunits of pertussis toxin. Although the CDTs are generally considered to all function as bacterial genotoxins, the extent to which individual members of the CDTs employ similar mechanisms of cell surface binding, uptake, and trafficking within sensitive cells is poorly understood. Recently, data have begun to emerge suggesting differences in the molecular basis by which individual CDTs interact with and enter host cells, suggesting the possibility that CDTs possess properties reflecting the specific niches idiosyncratic to those CDT bacterial pathogens that produce them. The extent to which functional differences between individual CDTs reflect the specific requirements for intoxicating cells and tissues within the diverse range of host microenvironments colonized by CDT-producing pathogenic bacteria remains to be experimentally explored.

  16. Eukaryotic translation initiation factor 5A2 promotes metabolic reprogramming in hepatocellular carcinoma cells.

    Science.gov (United States)

    Cao, Ting-Ting; Lin, Shu-Hai; Fu, Li; Tang, Zhi; Che, Chi-Ming; Zhang, Li-Yi; Ming, Xiao-Yan; Liu, Teng-Fei; Tang, Xu-Ming; Tan, Bin-Bin; Xiang, Di; Li, Feng; Chan, On-Yee; Xie, Dan; Cai, Zongwei; Guan, Xin-Yuan

    2017-01-01

    Reprogramming of intracellular metabolism is common in liver cancer cells. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. In our previous study, we reported that a novel oncogene eukaryotic translation initiation factor 5A2 (EIF5A2) promotes tumorigenesis under hypoxic condition. Here, we aim to investigate the role of EIF5A2 in cell metabolic reprogramming during hepatocellular carcinoma (HCC) development. In this study, we reported that the messenger RNA (mRNA) level of EIF5A2 was upregulated in 59 of 105 (56.2%) HCC clinical samples (P = 0.015), and EIF5A2 overexpression was significantly associated with shorter survival time of patients with HCC (P = 0.021). Ectopic expression of EIF5A2 in HCC cell lines significantly promoted cell growth and accelerated glucose utilization and lipogenesis rates. The high rates of glucose uptake and lactate secretion conferred by EIF5A2 revealed an abnormal activity of aerobic glycolysis in HCC cells. Several key enzymes involved in glycolysis including glucose transporter type 1 and 2, hexokinase 2, phosphofructokinase liver type, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase M2 isoform, phosphoglycerate mutase 1 and lactate dehydrogenase A were upregulated by overexpression of EIF5A2. Moreover, EIF5A2 showed positive correlations with FASN and ACSS2, two key enzymes involved in the fatty acid de novo biosynthetic pathway, at both protein and mRNA levels in HCC. These results indicated that EIF5A2 may regulate fatty acid de novo biosynthesis by increasing the uptake of acetate. In conclusion, our findings demonstrate that EIF5A2 has a critical role in HCC cell metabolic reprogramming and may serve as a prominent novel therapeutic target for liver cancer treatment. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Fluorescence turn-on detection of Sn2+ in live eukaryotic and prokaryotic cells.

    Science.gov (United States)

    Lan, Haichuang; Wen, Ying; Shi, Yunming; Liu, Keyin; Mao, Yueyuan; Yi, Tao

    2014-10-21

    Sn(2+) is usually added to toothpaste to prevent dental plaque and oral disease. However, studies of its physiological role and bacteriostatic mechanism are restricted by the lack of versatile Sn(2+) detection methods applicable to live cells, including Streptococcus mutans. Here we report two Sn(2+) fluorescent probes containing a rhodamine B derivative as a fluorophore, linked via the amide moiety to N,N-bis(2-hydroxyethyl)ethylenediamine (R1) and tert-butyl carbazate group (R2), respectively. These probes can selectively chelate Sn(2+) and show marked fluorescence enhancement due to the ring open reaction of rhodamine induced by Sn(2+) chelation. The probes have high sensitivity and selectivity for Sn(2+) in the presence of various relevant metal ions. Particularly, both R1 and R2 can target lysosomes, and R2 can probe Sn concentrations in lysosomes with rather acidic microenvironment. Furthermore, these two probes have low toxicity and can be used as imaging probes for monitoring Sn(2+) not only in live KB cells (eukaryotic) but also in Streptococcus mutans cells (prokaryotic), which is a useful tool to study the physiological function of Sn(2+) in biological systems.

  18. Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Vijayachandran, Lakshmi Sumitra; Viola, Cristina; Garzoni, Frederic; Trowitzsch, Simon; Bieniossek, Christoph; Chaillet, Maxime; Schaffitzel, Christiane; Busso, Didier; Romier, Christophe; Poterszman, Arnaud; Richmond, Timothy J; Berger, Imre

    2011-08-01

    Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

  19. The eukaryotic cell originated in the integration and redistribution of hyperstructures from communities of prokaryotic cells based on molecular complementarity.

    Science.gov (United States)

    Norris, Vic; Root-Bernstein, Robert

    2009-06-04

    In the "ecosystems-first" approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entailing the loss of redundant hyperstructures, the uncoupling of transcription and translation, and the emergence of introns and multiple chromosomes. Molecular complementarity also facilitated integration of bacterial hyperstructures to perform cytoskeletal and movement functions.

  20. Unicellular eukaryotes as models in cell and molecular biology: critical appraisal of their past and future value.

    Science.gov (United States)

    Simon, Martin; Plattner, Helmut

    2014-01-01

    Unicellular eukaryotes have been appreciated as model systems for the analysis of crucial questions in cell and molecular biology. This includes Dictyostelium (chemotaxis, amoeboid movement, phagocytosis), Tetrahymena (telomere structure, telomerase function), Paramecium (variant surface antigens, exocytosis, phagocytosis cycle) or both ciliates (ciliary beat regulation, surface pattern formation), Chlamydomonas (flagellar biogenesis and beat), and yeast (S. cerevisiae) for innumerable aspects. Nowadays many problems may be tackled with "higher" eukaryotic/metazoan cells for which full genomic information as well as domain databases, etc., were available long before protozoa. Established molecular tools, commercial antibodies, and established pharmacology are additional advantages available for higher eukaryotic cells. Moreover, an increasing number of inherited genetic disturbances in humans have become elucidated and can serve as new models. Among lower eukaryotes, yeast will remain a standard model because of its peculiarities, including its reduced genome and availability in the haploid form. But do protists still have a future as models? This touches not only the basic understanding of biology but also practical aspects of research, such as fund raising. As we try to scrutinize, due to specific advantages some protozoa should and will remain favorable models for analyzing novel genes or specific aspects of cell structure and function. Outstanding examples are epigenetic phenomena-a field of rising interest.

  1. Autophagy in unicellular eukaryotes

    NARCIS (Netherlands)

    Kiel, J.A.K.W.

    2010-01-01

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components

  2. Mechanisms of mitochondrial response to variations in energy demand in eukaryotic cells.

    Science.gov (United States)

    Devin, Anne; Rigoulet, Michel

    2007-01-01

    This review focuses on the different mechanisms involved in the adjustment of mitochondrial ATP production to cellular energy demand. The oxidative phosphorylation steady state at constant mitochondrial enzyme content can vary in response to energy demand. However, such an adaptation is tightly linked to a modification in both oxidative phosphorylation yield and phosphate potential and is obviously very limited in eukaryotic cells. We describe the three main mechanisms involved in mitochondrial response to energy demand. In heart cells, a short-term adjustment can be reached mainly through metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer and signal transmission. In such a complex regulatory mechanism, Ca(2+) signaling participates in activation of matricial dehydrogenases as well as mitochondrial ATP synthase. These processes allow a large increase in ATP production rate without an important modification in thermodynamic forces. For a long-term adaptation, two main mechanisms are involved: modulation of the mitochondrial enzyme content as a function of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of some respiratory chain complex subunits. Regardless of the mechanism involved (kinetic regulation by covalent modification or adjustment of mitochondrial enzyme content), the cAMP signaling pathway plays a major role in molecular signaling, leading to the mitochondrial response. We discuss the energetic advantages of these mechanisms.

  3. A three-dimensional finite element model of an adherent eukaryotic cell

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    McGarry J. G.

    2004-04-01

    Full Text Available Mechanical stimulation is known to cause alterations in the behaviour of cells adhering to a substrate. The mechanisms by which forces are transduced into biological responses within the cell remain largely unknown. Since cellular deformation is likely involved, further understanding of the biomechanical origins of alterations in cellular response can be aided by the use of computational models in describing cellular structural behaviour and in determining cellular deformation due to imposed loads of various magnitudes. In this paper, a finite element modelling approach that can describe the biomechanical behaviour of adherent eukaryotic cells is presented. It fuses two previous modelling approaches by incorporating, in an idealised geometry, all cellular components considered structurally significant, i.e. prestressed cytoskeleton, cytoplasm, nucleus and membrane components. The aim is to determine if we can use this model to describe the non-linear structural behaviour of an adherent cell and to determine the contribution of the various cellular components to cellular stability. Results obtained by applying forces (in the picoNewton range to the model membrane nodes suggest a key role for the cytoskeleton in determining cellular stiffness. The model captures non-linear structural behaviours such as strain hardening and prestress effects (in the region of receptor sites, and variable compliance along the cell surface. The role of the cytoskeleton in stiffening a cell during the process of cell spreading is investigated by applying forces to five increasingly spread cell geometries. Parameter studies reveal that material properties of the cytoplasm (elasticity and compressibility also have a large influence on cellular stiffness. The computational model of a single cell developed here is proposed as one that is sufficiently complex to capture the non-linear behaviours of the cell response to forces whilst not being so complex that the parameters

  4. Assessment of in vivo and in vitro genotoxicity of glibenclamide in eukaryotic cells.

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    Juliane Rocha de Sant'Anna

    Full Text Available Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.

  5. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Shan Qin; Hong Tang; Lian-San Zhao; Fang He; Yong Lin; Li Liu; Xiao-Mei He

    2003-01-01

    AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV, or BamHI/EcoR Ⅰ followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSGSUTPL/Flag plasmid vectors with T4DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preSl-preS2S and preSlS encoding genes,determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins.Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.CONCLUSION: Eight recombinant plasmids expressing S,M, L or preSlS proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells

  6. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  7. Damage of eukaryotic cells by the pore-forming toxin sticholysin II: Consequences of the potassium efflux.

    Science.gov (United States)

    Cabezas, Sheila; Ho, Sylvia; Ros, Uris; Lanio, María E; Alvarez, Carlos; van der Goot, F Gisou

    2017-02-04

    Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.

  8. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0.

    Science.gov (United States)

    Zhang, Lei; Zhao, Xihua; Zhang, Guoxiu; Zhang, Jiajia; Wang, Xuedong; Zhang, Suping; Wang, Wei; Wei, Dongzhi

    2016-02-09

    Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as 'LML 3.0', and an on-off control protocol of NHEJ pathway called 'OFN 1.0', using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.

  9. A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

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    Melanie L. Hutton

    2017-06-01

    Full Text Available The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248 with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01. HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05, and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05. Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

  10. Synthesis, characterization and in vitro assessment of the magnetic chitosan-carboxymethylcellulose biocomposite interactions with the prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Grumezescu, Alexandru Mihai; Andronescu, Ecaterina; Ficai, Anton; Bleotu, Coralia; Mihaiescu, Dan Eduard; Chifiriuc, Mariana Carmen

    2012-10-15

    Preparation and characterization of CS/Fe(3)O(4)/CMC composite scaffolds including the morphology, crystallinity, and the in vitro efficacy as antibiotic delivery vehicles as well as their influence on the eukaryotic cells are reported. The results demonstrated that the magnetic polymeric composite scaffolds are exhibiting structural and functional properties that recommend them for further applications in the biomedical field. They improve the activity of currently used antibiotics belonging to penicillins, macrolides, aminoglycosides, rifampicines and quinolones classes, representing potential macromolecular carriers for these antimicrobial substances, to achieve extracellular and intracellular targets. The obtained systems are not cytotoxic and do not influence the eukaryotic HCT8 cell cycle, representing potential tools for the delivery of drugs in a safe, effective and less expensive manner.

  11. Endosymbiotic theories for eukaryote origin

    Science.gov (United States)

    Martin, William F.; Garg, Sriram; Zimorski, Verena

    2015-01-01

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe. PMID:26323761

  12. Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

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    Yongming Wang

    2014-03-01

    Full Text Available Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB and piggyBac (PB that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1, a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

  13. A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization.

    Science.gov (United States)

    Hutton, Melanie L; D'Costa, Kimberley; Rossiter, Amanda E; Wang, Lin; Turner, Lorinda; Steer, David L; Masters, Seth L; Croker, Ben A; Kaparakis-Liaskos, Maria; Ferrero, Richard L

    2017-01-01

    The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

  14. Recognition of extremophilic archaeal viruses by eukaryotic cells: a promising nanoplatform from the third domain of life.

    Science.gov (United States)

    Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur; Papathanasiou, Pavlos; Peng, Xu; Moghimi, Seyed Moein

    2016-11-28

    Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration of novel medical nanoplatforms. Here, we selected two archaeal viruses Sulfolobus monocaudavirus 1 (SMV1) and Sulfolobus spindle shaped virus 2 (SSV2) owing to their unique spindle shape, hyperthermostable and acid-resistant nature and studied their interaction with mammalian cells. Accordingly, we followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path for selective cell targeting. On internalization, both viruses localize to the lysosomal compartments. Neither SMV1, nor SSV2 induced any detrimental effect on cell morphology, plasma membrane and mitochondrial functionality. This is the first study demonstrating recognition of archaeal viruses by eukaryotic cells which provides good basis for future exploration of archaeal viruses in bioengineering and development of multifunctional vectors.

  15. Dynamic instability--a common denominator in prokaryotic and eukaryotic DNA segregation and cell division.

    Science.gov (United States)

    Fuesler, John A; Li, Hsin-Jung Sophia

    2012-12-01

    Dynamic instability is an essential phenomenon in eukaryotic nuclear division and prokaryotic plasmid R1 segregation. Although the molecular machines used in both systems differ greatly in composition, strong similarities and requisite nuances in dynamics and segregation mechanisms are observed. This brief examination of the current literature provides a functional comparison between prokaryotic and eukaryotic dynamically unstable filaments, specifically ParM and microtubules. Additionally, this mini-review should support the notion that any dynamically unstable filament could serve as the molecular machine driving DNA segregation, but these machines possess auxiliary features to adapt to temporal and spatial disparities in either system.

  16. Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

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    David M Truong

    2015-08-01

    Full Text Available Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme" and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have

  17. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  18. Megaphylogeny, cell body plans, adaptive zones: causes and timing of eukaryote basal radiations.

    Science.gov (United States)

    Cavalier-Smith, Thomas

    2009-01-01

    I discuss eukaryote megaphylogeny and the timing of major innovations in the light of multigene trees and the rarity of marine/freshwater evolutionary transitions. The first eukaryotes were aerobic phagotrophs, probably substratum-associated heterotrophic amoeboflagellates. The primary eukaryote bifurcation generated unikonts (ancestrally probably unicentriolar, with a conical microtubular [MT] cytoskeleton) and bikonts (ciliary transformation from anterior cilium to ancestrally gliding posterior cilium; cytoskeleton of ventral MT bands). Unikonts diverged into Amoebozoa with anterior cilia, lost when lobosan broad pseudopods evolved for locomotion, and Choanozoa with posterior cilium and filose pseudopods that became unbranched tentacles/microvilli in holozoa and eventually the choanoflagellate/choanocyte collar. Of choanozoan ancestry, animals evolved epithelia, fibroblasts, eggs, and sperm. Fungi and Ichthyosporea evolved walls. Bikonts, ancestrally with ventral grooves, include three adaptively divergent megagroups: Rhizaria (Retaria and Cercozoa, ancestrally reticulofilose soft-surfaced gliding amoeboflagellates), and the originally planktonic Excavata, and the corticates (Plantae and chromalveolates) that suppressed pseudopodia. Excavata evolved cilia-generated feeding currents for grooval ingestion; corticates evolved cortical alveoli and ciliary hairs. Symbiogenetic origin and transfers of chloroplasts stimulated an explosive radiation of corticates--hard to resolve on multigene trees--and opisthokonts, and ensuing Cambrian explosions of animals and protists. Plantae lost phagotrophy and multiply evolved walls and macroalgae. Apusozoa, with dorsal pellicle and ventral pseudopods, are probably the most divergent bikonts or related to opisthokonts. Eukaryotes probably originated 800-850 My ago. Amoebozoa, Apusozoa, Loukozoa, and Metamonada may be the only extant eukaryote phyla pre-dating Neoproterozoic snowball earth. New subphyla are established for

  19. A biocompatible micro cell culture chamber for culturing and on-line monitoring of Eukaryotic cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2006-01-01

    Visualisering af cellulære processer over længere tidsperioder har været besværliggjort af cellernes krav til varme, fugtighed og et fysiologisk pH balanceret medie. Fremskridt indenfor mikro teknologi har muliggjort fabrikation af miniaturiserede celle kultur anordninger der er i stand til...... at holde celler i live over længere tidsperioder I det foreliggende arbejde præsenteres et nyt perfusions baseret mikro celle dyrknings kultur kammer med integreret termisk overvågning og regulering. Kammeret opretholdt både dyrkning og on-line overvågning af både kræft celler såvel som stam celler over...... at dyrknings betingelserne i kammeret var sammenlignelige med dem i konventionelle celle kultur dyrknings flaske, hvis lys intensiteten på mikroskopet og omgivelserne blev minimeret mest muligt. Overflade modificeringer af den strukturelle fotoresist SU-8, der ofte bliver brugt til fabrikation af mikro kanaler...

  20. Expanding the eukaryotic genetic code

    Energy Technology Data Exchange (ETDEWEB)

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2017-02-28

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  1. Expanding the eukaryotic genetic code

    Science.gov (United States)

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2013-01-22

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  2. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    Science.gov (United States)

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-02-19

    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  3. An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

    Science.gov (United States)

    Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

    2012-07-01

    cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

  4. Intra-plastid protein trafficking; how plant cells adapted prokaryotic mechanisms to the eukaryotic condition

    OpenAIRE

    Celedon, Jose M.; Cline, Kenneth

    2012-01-01

    Protein trafficking and localization in plastids involves a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to th...

  5. Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins

    OpenAIRE

    Poledňák, Jan

    2015-01-01

    The bacterial protein toxins endowed with the ability to translocate across the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eukaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transferring other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with other artificially prepared agents on synthetic membranes it allows detaile...

  6. Intra-plastid protein trafficking; how plant cells adapted prokaryotic mechanisms to the eukaryotic condition

    OpenAIRE

    Celedon, Jose M.; Cline, Kenneth

    2012-01-01

    Protein trafficking and localization in plastids involves a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to th...

  7. Intra-plastid protein trafficking; how plant cells adapted prokaryotic mechanisms to the eukaryotic condition

    Science.gov (United States)

    Celedon, Jose M.; Cline, Kenneth

    2012-01-01

    Protein trafficking and localization in plastids involves a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called ‘conservative sorting’. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. PMID:22750312

  8. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    Science.gov (United States)

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  9. [Genomic noncoding sequences and the size of eukaryotic cell nucleus as important factors of gene protection from chemical mutagens].

    Science.gov (United States)

    Minkevich, I G; Patrushev, L I

    2007-01-01

    An improved quantitative model describing a protective function of eukaryotic genomic noncoding sequences was developed. In this new model, two factors affecting gene protection from chemical mutagens are considered: (1) the ratio of the total lengths of coding and noncoding genomic sequences and (2) the volume of the cell nucleus. An increase in the noncoding DNA in the genome reduces the number of mutagen-damaged nucleotides in the coding region, whereas an increase in the volume of the nucleus decreases the flow of mutagens per unit of nuclear volume that attacks its surface.

  10. Synthesis, spectroscopic, and photophysical characterization and photosensitizing activity toward prokaryotic and eukaryotic cells of porphyrin-magainin and -buforin conjugates.

    Science.gov (United States)

    Dosselli, Ryan; Ruiz-González, Rubén; Moret, Francesca; Agnolon, Valentina; Compagnin, Chiara; Mognato, Maddalena; Sella, Valentina; Agut, Montserrat; Nonell, Santi; Gobbo, Marina; Reddi, Elena

    2014-02-27

    Cationic antimicrobial peptides (CAMPs) and photodynamic therapy (PDT) are attractive tools to combat infectious diseases and to stem further development of antibiotic resistance. In an attempt to increase the efficiency of bacteria inactivation, we conjugated a PDT photosensitizer, cationic or neutral porphyrin, to a CAMP, buforin or magainin. The neutral and hydrophobic porphyrin, which is not photoactive per se against Gram-negative bacteria, efficiently photoinactivated Escherichia coli after conjugation to either buforin or magainin. Conjugation to magainin resulted in the considerable strengthening of the cationic and hydrophilic porphyrin's interaction with the bacterial cells, as shown by the higher bacteria photoinactivation activity retained after washing the bacterial suspension. The porphyrin-peptide conjugates also exhibited strong interaction capability as well as photoactivity toward eukaryotic cells, namely, human fibroblasts. These findings suggest that these CAMPs have the potential to carry drugs and other types of cargo inside mammalian cells similar to cell-penetrating peptides.

  11. A factor converting viable but nonculturable Vibrio cholerae to a culturable state in eukaryotic cells is a human catalase.

    Science.gov (United States)

    Senoh, Mitsutoshi; Hamabata, Takashi; Takeda, Yoshifumi

    2015-08-01

    In our previous work, we demonstrated that viable but nonculturable (VBNC) Vibrio cholerae O1 and O139 were converted to culturable by coculture with eukaryotic cells. Furthermore, we isolated a factor converting VBNC V. cholerae to culturable (FCVC) from a eukaryotic cell line, HT-29. In this study, we purified FCVC by successive column chromatographies comprising UNO Q-6 anion exchange, Bio-Scale CHT2-1 hydroxyapatite, and Superdex 200 10/300 GL. Homogeneity of the purified FCVC was demonstrated by SDS-PAGE. Nano-LC MS/MS analysis showed that the purified FCVC was a human catalase. An experiment of RNAi knockdown of catalase mRNA from HT-29 cells and treatment of the purified FCVC with a catalase inhibitor, 3-amino-1,2,4-triazole confirmed that the FCVC was a catalase. A possible role of the catalase in converting a VBNC V. cholerae to a culturable state in the human intestine is discussed.

  12. Compositions and methods for the expression of selenoproteins in eukaryotic cells

    Science.gov (United States)

    Gladyshev, Vadim [Lincoln, NE; Novoselov, Sergey [Puschino, RU

    2012-09-25

    Recombinant nucleic acid constructs for the efficient expression of eukaryotic selenoproteins and related methods for production of recombinant selenoproteins are provided. The nucleic acid constructs comprise novel selenocysteine insertion sequence (SECIS) elements. Certain novel SECIS elements of the invention contain non-canonical quartet sequences. Other novel SECIS elements provided by the invention are chimeric SECIS elements comprising a canonical SECIS element that contains a non-canonical quartet sequence and chimeric SECIS elements comprising a non-canonical SECIS element that contains a canonical quartet sequence. The novel SECIS elements of the invention facilitate the insertion of selenocysteine residues into recombinant polypeptides.

  13. Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba Ⅰ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP Ⅱ for Uroplakin Ⅱ was successfully constructed.After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

  14. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...

  15. Replication protein A and more: single-stranded DNA-binding proteins in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Ting Liu; Jun Huang

    2016-01-01

    Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication,recombinational repair,and maintenance of genome stability.In human,the major SSB,replication protein A (RPA),is a stable heterotrimer composed of subunits of RPA1,RPA2,and RPA3,each df which is conserved not only in mammals but also in all other eukaryotic species.In addition to RPA,other SSBs have also been identified in the human genome,including sensor of single-stranded DNA complexes 1 and 2 (SOSS1/2).In this review,we summarize our current understanding of how these SSBs contribute to the maintenance of genome stability.

  16. Construction of Antibacterial Peptide CecropinB Eukaryotic Recombinant Vector and Its Expression in Dairy Goat Mammary Gland Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    GAO Xuejun; TONG Huili; YIN Deyun; ZHANG Li

    2008-01-01

    To investigate the expression of antibacterial peptide CecropinB eDNA in dairy goat mammary gland epithelial cells, the CecropinB gene was eloned and was inserted into a eukaryotic vector pECFP-Cl to construct the recombinant plasmid pECFP-B by genetic engineering technique. Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CeeropinB. The expression of CecropinB was also detected. The result of RT-PCR demonstrated CecropinB gene was expressed in transfeeted cells. CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland, exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.

  17. Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Song Gu; Chang-Jian Liu; Tong Qiao; Xue-Mei Sun; Lei-Lei Chen; Le Zhang

    2004-01-01

    AIM: To construct antisense VEGF165 eukaryotic expression vector PCDNA3-as-VEGF165 and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS: VEGF165 cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA3 to construct PCDNA3-as-VEGF165. Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.RESULTS: The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA3-as-VEGF165 transfection. The expression of VEGF protein was dramatically inhibited (142.01±7.95 vs 1 625.52±64.46 pg·ml-1, P<0.01) 2 days after transfection,which correlated with the dose of PCDNA3-as-VEGF165 gene.VEGF protein was most expressed in PCDNA3 transferred SMMC-7721 cells but few in PCDNA3-as-VEGF165 transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98±0.86% vs4.86±0.27%, P<0.01) and the survival rate was notably decreased (80.99±3.20% vs 93.52±3.93%, P<0.05) due to antisense VEGF165 by flow cytometry (FCM). The transfection of antisense VEGF165 gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.CONCLUSION: It is confirmed that antisense VEGF165 can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF165 gene therapy may play an important role in the treatment of human hepatocarcinoma.

  18. Thiopental inhibits global protein synthesis by repression of eukaryotic elongation factor 2 and protects from hypoxic neuronal cell death.

    Directory of Open Access Journals (Sweden)

    Christian I Schwer

    Full Text Available Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.

  19. 真核生物翻译过程中的mRNA质量控制%Quality Control of Eukaryotic mRNA in The Process of Translation

    Institute of Scientific and Technical Information of China (English)

    谢兆辉; 曾强成; 沈亮; 王继有

    2013-01-01

    成熟mRNA的合成是一个复杂的过程,往往会产生错误.原核和真核细胞都在多水平进化出了mRNA监视机制,以保证mRNA的质量,甚至在翻译起始之后.真核生物胞质中有4种翻译依赖性的mRNA质量监视机制:无意义介导的降解、No-go降解、Non-stop降解和核糖体延伸介导的降解.这些机制不仅可以识别并迅速降解有缺陷的mRNA,控制mRNA质量,还都在调节基因表达方面具有重要作用,而且也与一些遗传病有关.本文主要综述了真核生物4种mRNA质量监视机制的研究进展,并对相关研究的应用前景做了展望.%Production of mature mRNA consists of a highly complex pathway of synthesis, and errors often happen. Both prokaryotic and eukaryotic cells have evolved remarkable surveillance mechanisms acting at several steps of mRNA biogenesis, even after translation initiation, to control mRNA quality. In eukaryotic cells, there are 4 translation-dependent mRNA surveillance pathways in cytoplasm, including nonsense-mediated decay (NMD), no-go decay (NGD), non-stop decay (NSD) and ribosome extension-mediated decay (REMD). These mRNA surveillance systems not only contribute to recognize and rapidly degrades aberrant mRNAs, but also play an essential role in gene regulation, and associated with several human diseases. In this review, recent achievements in the investigation of eukaryotic mRNA surveillance pathways will be discussed, and their application perspective will also be speculated.

  20. L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase from a plant cultured cells, Nicotina tabacum L.

    Science.gov (United States)

    Kuriyama, Isoko; Asano, Naoki; Kato, Ikuo; Oshige, Masahiko; Sugino, Akio; Kadota, Yasuhiro; Kuchitsu, Kazuyuki; Yoshida, Hiromi; Sakaguchi, Kengo; Mizushina, Yoshiyuki

    2004-03-01

    We found a novel inhibitor specific to eukaryotic DNA polymerase epsilon(pol epsilon) from plant cultured cells, Nicotina tabacum L. The compound (compound 1) was a dipeptide alcohol, L-homoserylaminoethanol. The 50% inhibition of pol epsilon activity by the compound was 43.6 microg/mL, and it had almost no effect on the activities of the other eukaryotic DNA polymerases such as alpha, beta, gamma and delta, prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human telomerase, human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, human DNA topoisomerase I and II, T4 polynucleotide kinase and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of pol epsilon by the compound was non-competitive with respect to both template-primer DNA and nucleotide substrate. We succeeded in chemically synthesizing the stereoisomers, L-homoserylaminoethanol and D-homoserylaminoethanol, and found both were effective to the same extent. The IC(50) values of L- and D-homoserylaminoethanols for pol epsilon were 42.0 and 41.5 microg/mL, respectively. This represents the second discovery of a pol epsilon-specific inhibitor, and the first report on a water-soluble peptide-like compound as the inhibitor, which is required in biochemical studies of pol epsilon.

  1. Early evolution of eukaryote feeding modes, cell structural diversity, and classification of the protozoan phyla Loukozoa, Sulcozoa, and Choanozoa.

    Science.gov (United States)

    Cavalier-Smith, Thomas

    2013-05-01

    I discuss how different feeding modes and related cellular structures map onto the eukaryote evolutionary tree. Centrally important for understanding eukaryotic cell diversity are Loukozoa: ancestrally biciliate phagotrophic protozoa possessing a posterior cilium and ventral feeding groove into which ciliary currents direct prey. I revise their classification by including all anaerobic Metamonada as a subphylum and adding Tsukubamonas. Loukozoa, often with ciliary vanes, are probably ancestral to all protozoan phyla except Euglenozoa and Percolozoa and indirectly to kingdoms Animalia, Fungi, Plantae, and Chromista. I make a new protozoan phylum Sulcozoa comprising subphyla Apusozoa (Apusomonadida, Breviatea) and Varisulca (Diphyllatea; Planomonadida, Discocelida, Mantamonadida; Rigifilida). Understanding sulcozoan evolution clarifies the origins from them of opisthokonts (animals, fungi, Choanozoa) and Amoebozoa, and their evolutionary novelties; Sulcozoa and their descendants (collectively called podiates) arguably arose from Loukozoa by evolving posterior ciliary gliding and pseudopodia in their ventral groove. I explain subsequent independent cytoskeletal modifications, accompanying further shifts in feeding mode, that generated Amoebozoa, Choanozoa, and fungi. I revise classifications of Choanozoa, Conosa (Amoebozoa), and basal fungal phylum Archemycota. I use Choanozoa, Sulcozoa, Loukozoa, and Archemycota to emphasize the need for simply classifying ancestral (paraphyletic) groups and illustrate advantages of this for understanding step-wise phylogenetic advances.

  2. Structural organization of very small chromosomes: study on a single-celled evolutionary distant eukaryote Giardia intestinalis.

    Science.gov (United States)

    Tůmová, Pavla; Uzlíková, Magdalena; Wanner, Gerhard; Nohýnková, Eva

    2015-03-01

    During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.

  3. Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells.

    Science.gov (United States)

    Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen; Schneider, Robert J

    2015-08-01

    Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity.

  4. Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall

    Directory of Open Access Journals (Sweden)

    Neha eMalhotra

    2016-02-01

    Full Text Available Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB and PknL, Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behaviour. On the other hand, phosphomimics of mPDE (T309D or T309E, exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue.

  5. Chromosome segregation and organization are targets of 5'-Fluorouracil in eukaryotic cells.

    Science.gov (United States)

    Mojardín, Laura; Botet, Javier; Moreno, Sergio; Salas, Margarita

    2015-01-01

    The antimetabolite 5'-Fluorouracil (5FU) is an analog of uracil commonly employed as a chemotherapeutic agent in the treatment of a range of cancers including colorectal tumors. To assess the cellular effects of 5FU, we performed a genome-wide screening of the haploid deletion library of the eukaryotic model Schizosaccharomyces pombe. Our analysis validated previously characterized drug targets including RNA metabolism, but it also revealed unexpected mechanisms of action associated with chromosome segregation and organization (post-translational histone modification, histone exchange, heterochromatin). Further analysis showed that 5FU affects the heterochromatin structure (decreased levels of histone H3 lysine 9 methylation) and silencing (down-regulation of heterochromatic dg/dh transcripts). To our knowledge, this is the first time that defects in heterochromatin have been correlated with increased cytotoxicity to an anticancer drug. Moreover, the segregation of chromosomes, a process that requires an intact heterochromatin at centromeres, was impaired after drug exposure. These defects could be related to the induction of genes involved in chromatid cohesion and kinetochore assembly. Interestingly, we also observed that thiabendazole, a microtubule-destabilizing agent, synergistically enhanced the cytotoxic effects of 5FU. These findings point to new targets and drug combinations that could potentiate the effectiveness of 5FU-based treatments.

  6. Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate-chitosan-silica biocomposite.

    Science.gov (United States)

    Balaure, Paul Catalin; Andronescu, Ecaterina; Grumezescu, Alexandru Mihai; Ficai, Anton; Huang, Keng-Shiang; Yang, Chih-Hui; Chifiriuc, Carmen Mariana; Lin, Yung-Sheng

    2013-01-30

    This work is focused on the fabrication of a new drug delivery system based on polyanionic matrix (e.g. sodium alginate), polycationic matrix (e.g. chitosan) and silica network. The FT-IR, SEM, DTA-TG, eukaryotic cell cycle and viability, and in vitro assay of the influence of the biocomposite on the efficacy of antibiotic drugs were investigated. The obtained results demonstrated the biocompatibility and the ability of the fabricated biocomposite to maintain or improve the efficacy of the following antibiotics: piperacillin-tazobactam, cefepime, piperacillin, imipenem, gentamicin, ceftazidime against Pseudomonas aeruginosa ATCC 27853 and cefazolin, cefaclor, cefuroxime, ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole against Escherichia coli ATCC 25922 reference strains.

  7. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  8. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

    Science.gov (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail

    2015-10-01

    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  9. The spectrin-ankyrin-4.1-adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life.

    Science.gov (United States)

    Baines, Anthony J

    2010-08-01

    The cells in animals face unique demands beyond those encountered by their unicellular eukaryotic ancestors. For example, the forces engendered by the movement of animals places stresses on membranes of a different nature than those confronting free-living cells. The integration of cells into tissues, as well as the integration of tissue function into whole animal physiology, requires specialisation of membrane domains and the formation of signalling complexes. With the evolution of mammals, the specialisation of cell types has been taken to an extreme with the advent of the non-nucleated mammalian red blood cell. These and other adaptations to animal life seem to require four proteins--spectrin, ankyrin, 4.1 and adducin--which emerged during eumetazoan evolution. Spectrin, an actin cross-linking protein, was probably the earliest of these, with ankyrin, adducin and 4.1 only appearing as tissues evolved. The interaction of spectrin with ankyrin is probably a prerequisite for the formation of tissues; only with the advent of vertebrates did 4.1 acquires the ability to bind spectrin and actin. The latter activity seems to allow the spectrin complex to regulate the cell surface accumulation of a wide variety of proteins. Functionally, the spectrin-ankyrin-4.1-adducin complex is implicated in the formation of apical and basolateral domains, in aspects of membrane trafficking, in assembly of certain signalling and cell adhesion complexes and in providing stability to otherwise mechanically fragile cell membranes. Defects in this complex are manifest in a variety of hereditary diseases, including deafness, cardiac arrhythmia, spinocerebellar ataxia, as well as hereditary haemolytic anaemias. Some of these proteins also function as tumor suppressors. The spectrin-ankyrin-4.1-adducin complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

  10. Stabilization of polymer lipid complexes prepared with lipids of lactic acid bacteria upon preservation and internalization into eukaryotic cells.

    Science.gov (United States)

    Alves, P; Hugo, A A; Szymanowski, F; Tymczyszyn, E E; Pérez, P F; Coelho, J F J; Simões, P N; Gómez-Zavaglia, A

    2014-11-01

    The physicochemical characterization of polymer liposome complexes (PLCs) prepared with lipids of lactic acid bacteria and poly(N,N-dimethylaminoethyl methacrylate) covalently bound to cholesterol (CHO-PDMAEMA) was carried out in an integrated approach, including their stability upon preservation and incorporation into eukaryotic cells. PLCs were prepared with different polymer:lipid molar ratios (0, 0.05 and 0.10). Zeta potential, particle size distribution and polydispersity index were determined. The optimal polymer:lipid ratio and the stability of both bare liposomes and PLCs were evaluated at 37 °C and at different pHs, as well as after storage at 4 °C, -80 °C and freeze-drying in the presence or absence of trehalose 250 mM. Internalization of PLCs by eukaryotic cells was assessed to give a complete picture of the system. Incorporation of CHO-PDMAEMA onto bacterial lipids (ratio 0.05 and 0.10) led to stabilization at 37 °C and pH 7. A slight decrease of pH led to their strong destabilization. Bacteria PLCs showed to be more stable than lecithin (LEC) PLCs (used for comparison) upon preservation at 4 and -80 °C. The harmful nature of the preservation processes led to a strong decrease in the stability of PLCs, bacterial formulations being more stable than LEC PLCs. The addition of trehalose to the suspension of liposomes stabilized LEC PLC and did not have effect on bacterial PLCs. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. Preparations with higher stability were the ones that showed a better cell-uptake. The nature of the lipid composition is determinant for the stability of PLCs. Lipids from lactic acid bacteria are composed of glycolipids and phospholipids like cardiolipin and phosphatidylglycerol. The presence of negatively charged lipids strongly improves the interaction with the positively charged CHO-PDMAEMA, thus stabilizing liposomes. In addition, glycolipids and

  11. [Construction of eukaryotic expressing vector of multiple myeloma mucin-1 and its expression in COS-7 cells in vitro].

    Science.gov (United States)

    Liu, Kun; Luo, Yun-Jiao; Liu, Yue-Bo; Yao, Jin; Yang, Hong; Mou, Hong; Huang, Gui-Yun; Zhang, You

    2009-08-01

    In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.

  12. Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells

    Science.gov (United States)

    Deplanche, Martine; Filho, Rachid Aref El-Aouar; Alekseeva, Ludmila; Ladier, Emilie; Jardin, Julien; Henry, Gwénaële; Azevedo, Vasco; Miyoshi, Anderson; Beraud, Laetitia; Laurent, Frederic; Lina, Gerard; Vandenesch, François; Steghens, Jean-Paul; Le Loir, Yves; Otto, Michael; Götz, Friedrich; Berkova, Nadia

    2015-01-01

    Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1–4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection.—Deplanche, M., Filho. R. A. E.–A., Alekseeva, L., Ladier, E., Jardin, J., Henry, G., Azevedo, V., Miyoshi, A., Beraud, L., Laurent, F., Lina, G., Vandenesch, F., Steghens, J.-P., Le Loir, Y., Otto, M., Götz, F., Berkova, N. Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells. PMID:25648996

  13. Construction and expression of the eukaryotic expressed plasmid of MIC3 gene from Toxoplasma gondii in IBRS-2 cells

    Institute of Scientific and Technical Information of China (English)

    Tao JIANG; Donglin ZHANG; Hao NIE; Baoan YAO; Junlong ZHAO

    2008-01-01

    The sequence encoding MIC3 was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the vector pMD18-T. The tar-get gene was subcloned into the eukaryotic vector pcDNA3.1 after the identification of pMD18-T-MIC3 by enzyme digesting, PCR amplification and sequencing. Then the target recombinant plasmids pcMIC3 were transfected into IBRS-2 cells, and the positive cells con-taining pcMIC3 plasmids were obtained under the selec-tion of G418. The expressed proteins from the positive cells were detected by SDS-PAGE, Western blot and ELISA. The results showed that the DNA sequence iden-tity was 99.9% between amplified MIC3 and that from GenBank. The molecular weight of the recombinant MIC3 protein with good immuno-activity was about 39.2 ku. These available data would lay the foundation for further studies on DNA vaccine against Toxoplasma gondii.

  14. Sensory transduction in eukaryotes : A comparison between Dictyosteliurn and vertebrate cells

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Janssens, Pim M.W.; Erneux, Christophe

    1991-01-01

    The organization of multicellular organisms depends on cell-cell communication. The signal molecules are often soluble components in the extracellular fluid, but also include odors and light. A large array of surface receptors is involved in the detection of these signals. Signals are then transduce

  15. Cell control report

    CERN Document Server

    2013-01-01

    Please note this is a Short Discount publication. This extensive report provides an essential overview of cells and their use as factory automation building blocks. The following issues are discussed in depth: Cell integration Cell software and standards Future technologies applied to cells Plus Cell control applications including: - rotary parts manufacturing - diesel engine component development - general cell control development at the General Electric Corporation - a vendor list.

  16. CONSTRUCTION OF ACTIVE RECOMBINANT CASPASE-3 EUKARYOTIC EXPRESSION PLA SMID AND EFFECT OF r-CASPASE-3 ON APOPTOSIS OF PANCREATIC CARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To construct active recombinant cas pa ses-3 gene(r-caspases-3)eukaryotic expression plasmid and observe the apoptos is inducing activity of r-caspase-3 in pancreatic carcinoma cells. Methods pcDNA3.1(+)/r-caspase-3 was constructed and pan creatic carcinoma cells(PC-Ⅱ)were transfected with the pcDNA3.1(+)/r-caspases -3 by liposomes(LipofectAMINE).The expression of r-Caspase-3 mRNA in pancreat ic carcinoma cells was detected by reverse transcription process of the polymera se chain reaction(RT-PCR), and the signs of apoptosis were examined in pancreat ic carcinoma cells by the methods of the DNA electrophoresis and flow cytometry analysis(FACS).Results The sequence inserted in pBlueSKM/r-Caspase-3 p lasmid was coincident with that of the r-caspases-3. The evaluation result of pcDNA3.1(+)/r-caspases-3 through enzyme cutting was correct. A 894bp strap was observed by RT-PCR after pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/r-caspases-3 by liposomes. No strap was found in control groups. A characteristic DNA ladder was observed in pancreatic carcinoma cells DNA elect r ophoresis, and transparent hypodiploid karyotype peak was found by FACS. Conclusion The plasmid of pcDNA3.1(+)/r-Caspase-3 was c onstructed successfully, the expression of r-Caspase-3 mRMA in pancreatic carc inoma cells was confirmed by RT-PCR, and pcDNA3.1(+)/r-Caspase-3 can induce a poptosis in pancreatic carcinoma cells.

  17. A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Marc W Schmid

    Full Text Available The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM, linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq. As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.

  18. Adapt or die: how eukaryotic cells respond to prolonged activation of the spindle assembly checkpoint.

    Science.gov (United States)

    Rossio, Valentina; Galati, Elena; Piatti, Simonetta

    2010-12-01

    Many cancer-treating compounds used in chemotherapies, the so-called antimitotics, target the mitotic spindle. Spindle defects in turn trigger activation of the SAC (spindle assembly checkpoint), a surveillance mechanism that transiently arrests cells in mitosis to provide the time for error correction. When the SAC is satisfied, it is silenced. However, after a variable amount of time, cells escape from the mitotic arrest, even if the SAC is not satisfied, through a process called adaptation or mitotic slippage. Adaptation weakens the killing properties of antimitotics, ultimately giving rise to resistant cancer cells. We summarize here the mechanisms underlying this process and propose a strategy to identify the factors involved using budding yeast as a model system. Inhibition of factors involved in SAC adaptation could have important therapeutic applications by potentiating the ability of antimitotics to cause cell death.

  19. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems.

    Science.gov (United States)

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-11-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    Xichun Han; Hong Zhang; Mingku Jia; Gang Han; Weidong Jiang

    2004-01-01

    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.

  1. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    XichunHan; HongZhang; MingkuJia; GangHan; WeidongJiang

    2004-01-01

    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453. Cellular & Molecular Immunology.

  2. Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A.

    Science.gov (United States)

    Hyvönen, Mervi T; Keinänen, Tuomo A; Khomutov, Maxim; Simonian, Alina; Vepsäläinen, Jouko; Park, Jong Hwan; Khomutov, Alex R; Alhonen, Leena; Park, Myung Hee

    2012-02-01

    The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.

  3. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  4. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    Science.gov (United States)

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  5. Exopolysaccharides produced by Lactobacillus and Bifidobacterium strains abrogate in vitro the cytotoxic effect of bacterial toxins on eukaryotic cells.

    Science.gov (United States)

    Ruas-Madiedo, P; Medrano, M; Salazar, N; De Los Reyes-Gavilán, C G; Pérez, P F; Abraham, A G

    2010-12-01

    To evaluate the capability of the exopolysaccharides (EPS) produced by lactobacilli and bifidobacteria from human and dairy origin to antagonize the cytotoxic effect of bacterial toxins. The cytotoxicity of Bacillus cereus extracellular factors on Caco-2 colonocytes in the presence/absence of the EPS was determined by measuring the integrity of the tissue monolayer and the damage to the cell membrane (extracellular lactate dehydrogenase activity). Additionally, the protective effect of EPS against the haemolytic activity of the streptolysin-O was evaluated on rabbit erythrocytes. The EPS produced by Bifidobacterium animalis ssp. lactis A1 and IPLA-R1, Bifidobacterium longum NB667 and Lactobacillus rhamnosus GG were able to counteract the toxic effect of bacterial toxins on the eukaryotic cells at 1mg ml(-1) EPS concentration. The EPS A1 was the most effective in counteracting the effect of B. cereus toxins on colonocytes, even at lower doses (0·5mg ml(-1) ), whereas EPS NB667 elicited the highest haemolysis reduction on erythrocytes. The production of EPS by lactobacilli and bifidobacteria could antagonize the toxicity of bacterial pathogens, this effect being EPS and biological marker dependent. This work allows gaining insight about the mechanisms that probiotics could exert to improve the host health. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  6. Interactome and Gene Ontology provide congruent yet subtly different views of a eukaryotic cell

    Directory of Open Access Journals (Sweden)

    Marín Ignacio

    2009-07-01

    Full Text Available Abstract Background The characterization of the global functional structure of a cell is a major goal in bioinformatics and systems biology. Gene Ontology (GO and the protein-protein interaction network offer alternative views of that structure. Results This study presents a comparison of the global structures of the Gene Ontology and the interactome of Saccharomyces cerevisiae. Sensitive, unsupervised methods of clustering applied to a large fraction of the proteome led to establish a GO-interactome correlation value of +0.47 for a general dataset that contains both high and low-confidence interactions and +0.58 for a smaller, high-confidence dataset. Conclusion The structures of the yeast cell deduced from GO and interactome are substantially congruent. However, some significant differences were also detected, which may contribute to a better understanding of cell function and also to a refinement of the current ontologies.

  7. Microbeads and anchorage-dependent eukaryotic cells: the beginning of a new era in biotechnology.

    Science.gov (United States)

    Miller, A O; Menozzi, F D; Dubois, D

    1989-01-01

    Modern methods for the mass cultivation of anchorage-dependent mammalian cells started with the advent of microcarrier technology. Largely for reasons pertaining to their mode of preparation and ease of cultivation, 150-230 microns microbeads have been overwhelmingly adopted and the technology around them developed. To meet high biomass, macroporous microbeads have been developed. Also, the chemistry of the microsupport has been adapted in order to afford better protection of fragile cells to mechanical wear while simultaneously reorienting their differentiation towards the sought aims (production of cytokines, enzymes etc. ...). Future progress depends upon solutions being brought to problems inherent to this new technology (maintenance of steady state conditions of growth etc. ...) as well as to requirements arising from animal cell culture in general (biosensors, bioreactor's design etc. ...). Besides such technical implementations, biology at large is also expected to benefit from the advent of microcarriers in fields as diverse as the preparation of metaphasic chromosomes in bulk, toxicity testing, organ reconstitution following cell transplantation etc.

  8. The construction of cosmid libraries which can be used to transform eukaryotic cells.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); T. Lund; E.J. Murray; A.L. Mellor; H.H.M. Dahl; R.A. Flavell (Richard)

    1982-01-01

    textabstractCosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase g

  9. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells

    Directory of Open Access Journals (Sweden)

    George D Bittner

    2016-01-01

    Full Text Available The repair (sealing of plasmalemmal damage, consisting of small holes to complete transections, is critical for cell survival, especially for neurons that rarely regenerate cell bodies. We first describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current flow and dye barriers can, if appropriately used, provide more accurate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic oxidation on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion influx mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc. in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets, or in a single step involving the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG to seal cell membranes and rejoin severed axonal ends – an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures

  10. Regulation of mutagenesis by exogenous biological factors in the eukaryotic cell systems

    Directory of Open Access Journals (Sweden)

    Lukash L. L.

    2013-07-01

    Full Text Available The representations of the mutations and the nature of spontaneous mutation process and mutagenesis induced by exogenous oncoviruses, DNAs and proteins-mitogens are analysed. Exogenous biological factors induce DNA damages in regulatory-informational way, acting on the cellular systems for maintenance of genetical stability. Molecular mechanisms are the same as at spontaneous mutagenesis but they are realized with the participation of alien genetical material. Among biological mutagens, the oncoviruses and mobile genetic elements (MGEs are distinguished as the strongest destabilizing factors which direct tumor transformation of somatic mammalian cells. Genetical reprogramming or changing the programs of gene expression at the differentiation of stem and progenitor cells under growth factors and citokines is probably followed by mutations and recombinations as well.

  11. FACT is a sensor of DNA torsional stress in eukaryotic cells.

    Science.gov (United States)

    Safina, Alfiya; Cheney, Peter; Pal, Mahadeb; Brodsky, Leonid; Ivanov, Alexander; Kirsanov, Kirill; Lesovaya, Ekaterina; Naberezhnov, Denis; Nesher, Elimelech; Koman, Igor; Wang, Dan; Wang, Jianming; Yakubovskaya, Marianna; Winkler, Duane; Gurova, Katerina

    2017-02-28

    Transitions of B-DNA to alternative DNA structures (ADS) can be triggered by negative torsional strain, which occurs during replication and transcription, and may lead to genomic instability. However, how ADS are recognized in cells is unclear. We found that the binding of candidate anticancer drug, curaxin, to cellular DNA results in uncoiling of nucleosomal DNA, accumulation of negative supercoiling and conversion of multiple regions of genomic DNA into left-handed Z-form. Histone chaperone FACT binds rapidly to the same regions via the SSRP1 subunit in curaxin-treated cells. In vitro binding of purified SSRP1 or its isolated CID domain to a methylated DNA fragment containing alternating purine/pyrimidines, which is prone to Z-DNA transition, is much stronger than to other types of DNA. We propose that FACT can recognize and bind Z-DNA or DNA in transition from a B to Z form. Binding of FACT to these genomic regions triggers a p53 response. Furthermore, FACT has been shown to bind to other types of ADS through a different structural domain, which also leads to p53 activation. Thus, we propose that FACT acts as a sensor of ADS formation in cells. Recognition of ADS by FACT followed by a p53 response may explain the role of FACT in DNA damage prevention. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

    DEFF Research Database (Denmark)

    Nielsen, Olaf; Løbner-Olesen, Anders

    2008-01-01

    by using the model organisms Escherichia coli and Schizosaccharomyces pombe as examples. Although the underlying molecular details are different, the logic behind the control mechanisms is similar. For example, after initiation, crucial molecules required for the loading of replicative helicases in both...

  13. In vivo Biotinylation Based Method for the Study of Protein-Protein Proximity in Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Arman Kulyyassov

    2014-01-01

    Full Text Available Introduction: The spatiotemporal order plays an important role in cell functioning and is affected in many pathologies such as cancer and neurodegenerative diseases. One of the ultimate goals of molecular biology is reconstruction of the spatiotemporal structure of a living cell at the molecular level. This task includes determination of proximities between different molecular components in the cell and monitoring their time- and physiological state-dependent changes. In many cases, proximity between macromolecules arises due to their interactions; however, the contribution of dynamic self-organization in generation of spatiotemporal order is emerging as another viable possibility. Specifically, in proteomics, this implies that the detection of protein-protein proximity is a more general task than gaining information about physical interactions between proteins, as it could detail aspects of spatial order in vivo that are challenging to reconstitute in binding experiments in vitro. Methods: In this work, we have developed a method of monitoring protein-protein proximity in vivo. For this purpose, the BirA was fused to one of the interaction partners, whereas the BAP was modified to make the detection of its biotinylation possible by mass spectrometry. Results: Using several experimental systems, we showed that the biotinylation is interaction dependent. In addition, we demonstrated that BAP domains with different primary amino acid structures and thus with different molecular weights can be used in the same experiment, providing the possibility of multiplexing. Alternatively to the changes in primary amino acid structure, the stable isotope format can also be used, providing another way to perform multiplexing experiments. Finally, we also demonstrated that our system could help to overcome another limitation of current methodologies to detect protein-protein proximity. For example, one can follow the state of a protein of interest at a defined

  14. Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells.

    Science.gov (United States)

    Faurez, Florence; Dory, Daniel; Henry, Aurélie; Bougeard, Stéphanie; Jestin, André

    2010-04-01

    In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes.

  15. Eukaryotic Cell Cycle as a Test Case for Modeling Cellular Regulation in a Collaborative Problem-Solving Environment

    Science.gov (United States)

    2007-03-01

    that encode the proteins that carry out the functions. Hence, we may think of the wild-type genome of 6 budding yeast as encoding a set of proteins...and in prokaryotes ( cyanobacteria ). In eukaryotes, there is a distinct negative feedback loop, whereby the period protein inhibits transcription of...rhythms in cyanobacteria are generated by a molecular mechanism that is much different from that in eukaryotes. The basic oscillatory proteins, KaiA-B

  16. ATP consumption of eukaryotic flagella measured at a single-cell level

    CERN Document Server

    Chen, Daniel T N; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-01-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating wavef...

  17. Cloning of the Eukaryotic Expression Vector with Nerve Growth Factor in Rats and Its Effects on Proliferation and Differentiation of Mesencephal Neural Stem Cells of Fetal Rats

    Institute of Scientific and Technical Information of China (English)

    Minhua LIN; Lin YANG; Rong FU; Hongyang ZHAO

    2008-01-01

    Summary: The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) β subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF β subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGE The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF β was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and faciliate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF β subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

  18. Mechanism of oxidative stress involved in the toxicity of ZnO nanoparticles against eukaryotic cells

    Directory of Open Access Journals (Sweden)

    M. Saliani

    2016-01-01

    Full Text Available ZnO NPs (zinc oxide nanoparticles has generated significant scientific interest as a novel antibacterial and anticancer agent. Since oxidative stress is a critical determinant of ZnO NPs-induced damage, it is necessary to characterize their underlying mode of action. Different structural and physicochemical properties of ZnO NPs such as particle surface, size, shape, crystal structure, chemical position, and presence of metals can lead to changes in biological activities including ROS (reactive oxygen species production. However, there are some inconsistencies in the literature on the relation between the physicochemical features of ZnO NPs and their plausible oxidative stress mechanism. Herein, the possible oxidative stress mechanism of ZnO NPs was reviewed. This is worthy of further detailed evaluations in order to improve our understanding of vital NPs characteristics governing their toxicity. Therefore, this study focuses on the different reported oxidative stress paradigms induced by ZnO NPs including ROS generated by NPs, oxidative stress due to the NPs-cell interaction, and role of the particle dissolution in the oxidative damage. Also, this study tries to characterize and understand the multiple pathways involved in oxidative stress induced by ZnO NPs. Knowledge about different cellular signaling cascades stimulated by ZnO NPs lead to the better interpretation of the toxic influences induced by the cellular and acellular parameters. Regarding the potential benefits of toxic effects of ZnO NPs, in-depth evaluation of their toxicity mechanism and various effects of these nanoparticles would facilitate their implementation for biomedical applications.

  19. Construction of Eukaryotic Expressing Plasmids Encoding HA and HA1 of Influenza A Virus and Their Transient Expression in HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Weidong; LI Mingyuan; CAO Kang; YANG Jing; SHI Qiaofa; WANG Baoning; JIANG Zhonghua; LI Hong

    2006-01-01

    In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 (+) to generate pcDNA3. 1 (+)/HA and pcDNA3.1 (+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1 (+)/HA and pcDNA3.1 (+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 (+)/HA or pcDNA3. 1 (+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

  20. Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process

    Science.gov (United States)

    Murphy, John R.

    2011-01-01

    Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. PMID:22069710

  1. Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system.

    Science.gov (United States)

    Taira, Hikaru; Fukushima, Masaharu; Hohsaka, Takahiro; Sisido, Masahiko

    2005-05-01

    Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.

  2. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part A: Eukaryotic Gene Structure and DNA Replication

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available Progress in the basic sciences of cell and molecular biology has provided an exciting dimension that has translated into clinically relevant information in every medical subspecialty. Importantly, the application of recombinant DNA technology has played a major role in unravelling the intricacies related to the molecular pathophysiology of disease. This series of review articles constitutes a framework for the integration of the database of new information into the core knowledge base of concepts related to the pathogenesis of gastrointestinal disorders and liver disease. The goal of this series of three articles is to review the basic principles of eukaryotic gene expression. The first article examines the role of DNA in directing the flow of genetic information in eukaryotic cells.

  3. Cell Control Engineering

    DEFF Research Database (Denmark)

    Lynggaard, Hans Jørgen Birk; Alting, Leo

    1996-01-01

    The engineering process of creating cell control systems is described, and a Cell Control Engineering (CCE) concept is defined. The purpose is to assist people, representing different disciplines in the organisation, to implement cell controllers by addressing the complexity of having many systems...... in physically and logically different and changing manufacturing environments. The defined CCE concept combines state-of-the-art of commercially available enabling technologies for automation system software development, generic cell control models and guidelines for the complete engineering process....... It facilitates the understanding of the task and structure of cell controllers and uses this knowledge directly in the implementation of the system. By applying generic models CCE facilitates reuse of software components and maintenance of applications. In many enterprises, software makes up an increasing part...

  4. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

  5. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

  6. Open Questions on the Origin of Eukaryotes.

    Science.gov (United States)

    López-García, Purificación; Moreira, David

    2015-11-01

    Despite recent progress, the origin of the eukaryotic cell remains enigmatic. It is now known that the last eukaryotic common ancestor was complex and that endosymbiosis played a crucial role in eukaryogenesis at least via the acquisition of the alphaproteobacterial ancestor of mitochondria. However, the nature of the mitochondrial host is controversial, although the recent discovery of an archaeal lineage phylogenetically close to eukaryotes reinforces models proposing archaea-derived hosts. We argue that, in addition to improved phylogenomic analyses with more comprehensive taxon sampling to pinpoint the closest prokaryotic relatives of eukaryotes, determining plausible mechanisms and selective forces at the origin of key eukaryotic features, such as the nucleus or the bacterial-like eukaryotic membrane system, is essential to constrain existing models.

  7. 原核和真核细胞表达HBeAg的应用研究%Application of HBeAg produced in prokaryotic cells and eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    何亮; 邓小昭; 刁振宇; 周宗安; 郑纪山; 高健

    2001-01-01

    Objectives: To express HBeAg in prokaryotic and eukaryotic cells and compare the two types of HBeAg in the anti-HBeAg testing. Methods: HBeAg was expressed both in E.coli cells and in silk worm cells, purified by Sephacryl S-200.HBeAg protein concentration and antigenic titer were determined respectively by ultraviolet-spectroscopy and EIA. Results: HBeAg produced by E.coli cells: Activation ratio was 10 000/mg, HBeAg/HBcAg = 50; The specificity in testing anti-HbeAg was 96%;HBeAg produced by silk worm cells: Activation ratio was 160 000/mg, HBeAg/HBcAg = 5 000, The specificity in testing anti-HbeAg was 100%. Conclusions: HBeAg produced by eukaryotic cells contained much lower proportion of HbcAg and higher activation ratio, which therefore bring about a possibility to improve the quality of the kit for testing Anti-HBe.%目的:将原核细胞和真核细胞分别克隆表达生产的HBeAg,经适当纯化后进行检测分析,并在乙型肝炎抗HBe检测中进行应用和比较。方法:分别用大肠杆菌和家蚕细胞表达生产HBeAg,并用Saphacryl S-200 柱层析进行纯化;紫外分光光度法测定表达产物的蛋白含量;EIA法测定HBeAg和HBcAg效价及评估HBeAg 的应用效果。结果:原核细胞HBeAg:比活性为10 000 /mg,HBeAg/HBcAg=50,用于抗HBe 的检测时特异性为96%,灵敏度符合国家卫生部panel要求。真核细胞HBeAg:比活性为 160 000/mg,HBeAg / HBcAg =5 000,用于抗HBe 的检测时特异性为100%,灵敏度高于国家卫生部panel要求的1~2个滴度。结论:真核细胞表达的HBeAg比活性高而HBcAg含量低,在抗HBe检测时的应用效果优于原核细胞。

  8. Noise in eukaryotic gene expression

    Science.gov (United States)

    Blake, William J.; KÆrn, Mads; Cantor, Charles R.; Collins, J. J.

    2003-04-01

    Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

  9. Eukaryotic vs. prokaryotic chemosensory systems.

    Science.gov (United States)

    Sbarbati, Andrea; Merigo, Flavia; Osculati, Francesco

    2010-04-01

    In the last decades, microbiologists demonstrated that microorganisms possess chemosensory capabilities and communicate with each other via chemical signals. In parallel, it was demonstrated that solitary eukaryotic chemosensory cells are diffusely located on the mucosae of digestive and respiratory apparatuses. It is now evident that on the mucosal surfaces of vertebrates, two chemoreceptorial systems (i.e. eukaryotic and prokaryotic) coexist in a common microenvironment. To date, it is not known if the two chemosensory systems reciprocally interact and compete for detection of chemical cues. This appears to be a fruitful field of study and future researches must consider that the mucosal epithelia possess more chemosensory capabilities than previously supposed.

  10. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  11. Abnormal Expression of Eukaryotic Translation Factors in Malignant Transformed Human Bronchial Epithelial Cells Induced by Crystalline Nickel Sulfide

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1δ (TEF-1δ) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS). Methods Abnormal expressions of human TIF3 and TEF-1δ genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT-PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively. Results RT-PCR analysis primarily showed that both human TIF3 and TEF-1δ mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-1δ cDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1δ genes were related to malignant degree of the cells induced by nickel. Conclusions These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1δ genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.

  12. Translational control of Arabidopsis meristem stability and organogenesis by the eukaryotic translation factor eIF3h.

    Directory of Open Access Journals (Sweden)

    Fujun Zhou

    Full Text Available Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3. Many of the mRNAs that function in apical meristem maintenance possess upstream open reading frames (uORFs, translational attenuators that render translation partially dependent on eIF3h. Specifically, the mRNA for the receptor kinase, CLV1, is undertranslated in the eif3h mutant as shown by transient and transgenic expression assays. Concordant phenotypic observations include defects in organ polarity and in translation of another uORF-containing mRNA, ASYMMETRIC LEAVES 1 (AS1, in eif3h. In summary, the expression of developmental regulatory mRNAs is attenuated by uORFs, and this attenuation is balanced in part by the translation initiation factor, eIF3h. Thus, translational control plays a key role in Arabidopsis stem cell regulation and organogenesis.

  13. Marked elevation of hypusine formation activity on eukaryotic initiation factor 5A in v-HA-RAS transformed mouse NIH3T3 cells.

    Science.gov (United States)

    Chen, Z P; Chen, K Y

    1997-05-19

    Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor is ubiquitously present in eukaryotic cells and archebacteria. In this reaction, deoxyhypusine synthase catalyzes the conversion of one unique lysine residue on eIF-5A to deoxyhypusine using spermidine as the substrate. Hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Hypusine formation activity can be measured by an in vitro labeling technique in polyamine-depleted cells. In addition, an in vitro cross-labeling assay can be employed to measure simultaneously the relative deoxyhypusine synthase activity and protein substrate amount. Using these approaches, together with Western blot analysis, we showed that hypusine formation activity is serum-responsive and significantly elevated in Ras oncogene transfected NIH3T3 cells as compared to NIH3T3 cells. The large difference, >30-fold, in hypusine formation activity between these two cells is mainly due to difference in the amount of newly synthesized eIF-5A precursor rather than deoxyhypusine synthase. The deoxyhypusine synthase activity is about three-fold higher in Ras-3T3 cells than in 3T3 cells, and remains constant throughout serum stimulation in both cells. Despite the significant difference in eIF-5A protein amounts, the eIF-5A mRNA levels in 3T3 cells and in Ras-3T3 cells are almost identical. Furthermore, unlike serum-dependent increase in eIF-5A precursor protein, the eIF-5A mRNA in both cells is constitutively expressed after serum stimulation, suggesting that eIF-5A gene is regulated at posttranscriptional/translational level during serum stimulation and cell transformation.

  14. The candidate phylum Poribacteria by single-cell genomics: new insights into phylogeny, cell-compartmentation, eukaryote-like repeat proteins, and other genomic features.

    Directory of Open Access Journals (Sweden)

    Janine Kamke

    Full Text Available The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.

  15. The candidate phylum Poribacteria by single-cell genomics: new insights into phylogeny, cell-compartmentation, eukaryote-like repeat proteins, and other genomic features.

    Science.gov (United States)

    Kamke, Janine; Rinke, Christian; Schwientek, Patrick; Mavromatis, Kostas; Ivanova, Natalia; Sczyrba, Alexander; Woyke, Tanja; Hentschel, Ute

    2014-01-01

    The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.

  16. The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features

    Science.gov (United States)

    Kamke, Janine; Rinke, Christian; Schwientek, Patrick; Mavromatis, Kostas; Ivanova, Natalia; Sczyrba, Alexander; Woyke, Tanja; Hentschel, Ute

    2014-01-01

    The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake. PMID:24498082

  17. [Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats].

    Science.gov (United States)

    Qiao, Li-Fen; Xu, Yong-Jian; Liu, Xian-Sheng; Xie, Jun-Gang; Du, Chun-Ling; Zhang, Jian; Ni, Wang; Chen, Shi-Xin

    2008-03-01

    This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis

  18. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Blake A Jacobson

    Full Text Available BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.

  19. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  20. Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells

    Directory of Open Access Journals (Sweden)

    Tao LI

    2011-04-01

    Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.

  1. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  2. Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO(2).

    Science.gov (United States)

    Saverini, Marghereth; Catanzaro, Irene; Sciandrello, Giulia; Avellone, Giuseppe; Indelicato, Sergio; Marcì, Giuseppe; Palmisano, Leonardo

    2012-03-01

    The presence of (±)α-pinene, (+)β-pinene, (+)3-carene, and R-(+)limonene terpenes in wastewater of a citrus transformation factory was detected and analyzed, in a previous study, by using Solid Phase Micro-extraction (SPME) followed by GC analyses. Purpose of that research was to compare the genotoxic responses of mixtures of terpenes with the genotoxicity of the individual compounds, and the biological effects of actual wastewater. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test) and in V79 cells by Comet assay. Ames tests indicated that the four single terpenes did not induce an increase of revertants frequency. On the contrary, the mixtures of terpenes caused, in the presence of metabolic activation, a highly significant increase of the revertants in TA100 strain in comparison to the control. The Comet assay showed a significant increase in DNA damage in V79 cells treated for 1h with single or mixed terpenes. Moreover, the actual wastewater was found highly genotoxic in bacterial and mammalian cells. Photocatalytic tests completely photodegraded the pollutants present in aqueous wastewater and the initial high genotoxicity of samples of wastewater collected during the photocatalytic run, was completely lose in 3h of irradiation.

  3. N1-guanyl-1,7-diaminoheptane sensitizes bladder cancer cells to doxorubicin by preventing epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2 activation.

    Science.gov (United States)

    Yang, Jinsong; Yu, Haogang; Shen, Mo; Wei, Wei; Xia, Lihong; Zhao, Peng

    2014-02-01

    Drug resistance greatly reduces the efficacy of doxorubicin-based chemotherapy in bladder cancer treatment; however, the underlying mechanisms are poorly understood. We aimed to investigate whether N1-guanyl-1,7-diaminoheptane (GC7), which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation, exerts synergistic cytotoxicity with doxorubicin in bladder cancer, and whether eIF5A2 is involved in chemoresistance to doxorubicin-based bladder cancer treatment. BIU-87, J82, and UM-UC-3 bladder cancer cells were transfected with eIF5A2 siRNA or negative control siRNA before incubation with doxorubicin alone or doxorubicin plus GC7 for 48 h. Doxorubicin cytotoxicity was enhanced by GC7 in BIU-87, J82, and UM-UC-3 cells. It significantly inhibited activity of eIF5A2, suppressed doxorubicin-induced epithelial-mesenchymal transition in BIU-87 cells, and promoted mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Knockdown of eIF5A2 sensitized bladder cancer cells to doxorubicin, prevented doxorubicin-induced EMT in BIU-87 cells, and encouraged mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Combination therapy with GC7 may enhance the therapeutic efficacy of doxorubicin in bladder cancer by inhibiting eIF5A2 activation and preventing epithelial-mesenchymal transition.

  4. The eukaryotic fossil record in deep time

    Science.gov (United States)

    Butterfield, N.

    2011-12-01

    Eukaryotic organisms are defining constituents of the Phanerozoic biosphere, but they also extend well back into the Proterozoic record, primarily in the form of microscopic body fossils. Criteria for identifying pre-Ediacaran eukaryotes include large cell size, morphologically complex cell walls and/or the recognition of diagnostically eukaryotic cell division patterns. The oldest unambiguous eukaryote currently on record is an acanthomorphic acritarch (Tappania) from the Palaeoproterozoic Semri Group of central India. Older candidate eukaryotes are difficult to distinguish from giant bacteria, prokaryotic colonies or diagenetic artefacts. In younger Meso- and Neoproterozoic strata, the challenge is to recognize particular grades and clades of eukaryotes, and to document their macro-evolutionary expression. Distinctive unicellular forms include mid-Neoproterozoic testate amoebae and phosphate biomineralizing 'scale-microfossils' comparable to an extant green alga. There is also a significant record of seaweeds, possible fungi and problematica from this interval, documenting multiple independent experiments in eukaryotic multicellularity. Taxonomically resolved forms include a bangiacean red alga and probable vaucheriacean chromalveolate algae from the late Mesoproterozoic, and populations of hydrodictyacean and siphonocladalean green algae of mid Neoproterozoic age. Despite this phylogenetic breadth, however, or arguments from molecular clocks, there is no convincing evidence for pre-Ediacaran metazoans or metaphytes. The conspicuously incomplete nature of the Proterozoic record makes it difficult to resolve larger-scale ecological and evolutionary patterns. Even so, both body fossils and biomarker data point to a pre-Ediacaran biosphere dominated overwhelming by prokaryotes. Contemporaneous eukaryotes appear to be limited to conspicuously shallow water environments, and exhibit fundamentally lower levels of morphological diversity and evolutionary turnover than

  5. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.

    2014-11-21

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  6. The viral eukaryogenesis hypothesis: a key role for viruses in the emergence of eukaryotes from a prokaryotic world environment.

    Science.gov (United States)

    Bell, Philip John Livingstone

    2009-10-01

    Understanding how the gulf between prokaryotic and eukaryotic cellular design arose is a major challenge. The viral eukaryogenesis (VE) hypothesis addresses the challenge of eukaryotic origins by suggesting the first eukaryotic cell was a multimember consortium consisting of a viral ancestor of the nucleus, an archaeal ancestor of the eukaryotic cytoplasm, and a bacterial ancestor of the mitochondria. Using only prokaryotes and their viruses, and invoking selective pressures observed in modern organisms, the VE hypothesis can explain the origins of the eukaryotic cell, sex, and meiosis. In the VE hypothesis, a cell wall-less archaeon and an alpha-proteobacterium established a syntrophic relationship, and then a complex DNA virus permanently lysogenized the archaeal syntroph to produce a consortium of three organisms that evolved into the eukaryotic cell. The mechanisms by which the virus replicated, controlled its copy number, and segregated to daughter cells led to the evolution of the asexual mitotic replication cycle and the sexual meiotic replication cycle. The VE hypothesis conceptually unifies prokaryotic and eukaryotic sex into variants of a single process.

  7. Transfer of DNA from Bacteria to Eukaryotes

    Directory of Open Access Journals (Sweden)

    Benoît Lacroix

    2016-07-01

    Full Text Available Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen, Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium, or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs, the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer.

  8. Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death.

    Science.gov (United States)

    Feng, Haizhong; Chen, Qingguo; Feng, Jian; Zhang, Jian; Yang, Xiaohui; Zuo, Jianru

    2007-07-01

    The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.

  9. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells.

    Science.gov (United States)

    Gaddy, Jennifer A; Tomaras, Andrew P; Actis, Luis A

    2009-08-01

    The ability of Acinetobacter baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to Candida albicans filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of A. baumannii 19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.

  10. Toxin Transport by A-B Type of Toxins in Eukaryotic Target Cells and Its Inhibition by Positively Charged Heterocyclic Molecules.

    Science.gov (United States)

    Benz, Roland; Barth, Holger

    2017-07-04

    A-B types of toxins are among the most potent bacterial protein toxins produced by gram-positive bacteria. Prominent examples are the tripartite anthrax toxin of Bacillus anthracis and the different A-B type clostridial toxins that are the causative agents of severe human and animal diseases and could serve as biological weapons. The components of all these toxins comprise one binding/transport (B) subunit and one or two separate, non-linked enzymatically active (A) subunits. The A and B subunits are separately produced and secreted by the pathogenic gram-positive bacteria and must assemble on the surface of eukaryotic target cells to form biologically active toxin complexes. The B components are cleaved by proteases to generate the biologically active species that binds to receptors on the surface of the target cells and form there oligomers which bind the A subunits. The AB complexes are internalized by receptor-mediated endocytosis and reach early or late endosomes that become acidified. Subsequently, the B components form channels in endosomal membranes that are indispensable for the transport of the enzymatic subunits across these membranes into the cytosol of target cells via the trans-membrane channels. In addition to the channels formed by the B components, host cell factors including chaperones and further folding helper enzymes are involved in the import of the enzymatic subunits into the cytosol of eukaryotic cells. Positively charged heterocyclic molecules, such as chloroquine and related aminoquinolinium and azolopyridinium salts have been shown in recent years to bind with high affinity to the channels formed by the B components of binary toxins. Since binding to the B components is also a prerequisite for transport of the A components across the endosomal membranes the channel blockers also prevent transport of the A subunits into the host cell cytosol. The inhibition of toxin uptake into cells by such pharmacological compounds should also be of

  11. Complex archaea that bridge the gap between prokaryotes and eukaryotes.

    Science.gov (United States)

    Spang, Anja; Saw, Jimmy H; Jørgensen, Steffen L; Zaremba-Niedzwiedzka, Katarzyna; Martijn, Joran; Lind, Anders E; van Eijk, Roel; Schleper, Christa; Guy, Lionel; Ettema, Thijs J G

    2015-05-14

    The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of 'Lokiarchaeota', a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic 'starter-kit' to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

  12. New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Laura Mojardín

    Full Text Available 5-Fluorouracil (5FU is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

  13. Evolution of viruses and cells: do we need a fourth domain of life to explain the origin of eukaryotes?

    Science.gov (United States)

    Moreira, David; López-García, Purificación

    2015-09-26

    The recent discovery of diverse very large viruses, such as the mimivirus, has fostered a profusion of hypotheses positing that these viruses define a new domain of life together with the three cellular ones (Archaea, Bacteria and Eucarya). It has also been speculated that they have played a key role in the origin of eukaryotes as donors of important genes or even as the structures at the origin of the nucleus. Thanks to the increasing availability of genome sequences for these giant viruses, those hypotheses are amenable to testing via comparative genomic and phylogenetic analyses. This task is made very difficult by the high evolutionary rate of viruses, which induces phylogenetic artefacts, such as long branch attraction, when inadequate methods are applied. It can be demonstrated that phylogenetic trees supporting viruses as a fourth domain of life are artefactual. In most cases, the presence of homologues of cellular genes in viruses is best explained by recurrent horizontal gene transfer from cellular hosts to their infecting viruses and not the opposite. Today, there is no solid evidence for the existence of a viral domain of life or for a significant implication of viruses in the origin of the cellular domains.

  14. Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue.

    Science.gov (United States)

    Park, M H; Liu, T Y; Neece, S H; Swiggard, W J

    1986-11-05

    Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

  15. Asgard archaea illuminate the origin of eukaryotic cellular complexity.

    Science.gov (United States)

    Zaremba-Niedzwiedzka, Katarzyna; Caceres, Eva F; Saw, Jimmy H; Bäckström, Disa; Juzokaite, Lina; Vancaester, Emmelien; Seitz, Kiley W; Anantharaman, Karthik; Starnawski, Piotr; Kjeldsen, Kasper U; Stott, Matthew B; Nunoura, Takuro; Banfield, Jillian F; Schramm, Andreas; Baker, Brett J; Spang, Anja; Ettema, Thijs J G

    2017-01-19

    The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the 'Asgard' superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of 'eukaryote-specific' proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.

  16. Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs.

    Science.gov (United States)

    Tome, M E; Gerner, E W

    1996-11-15

    Selection of HTC cells in drugs that inhibit ornithine decarboxylase (ODC) has produced two cell lines, HMOA and DH23A/b, that contain increased amounts of more stable ODC. In addition to alterations in ODC, these cells appear to produce modified eukaryotic initiation factor 5A (eIF-5A) at different rates, a reaction that both requires spermidine and is essential for proliferation. Alterations to the modification of eIF-5A by spermidine cannot be accounted for by changes in eIF-5A protein or modified eIF-5A turnover. Deoxyhypusine synthetase activity is similar in the parental and variant cell lines and is unaltered by growth into plateau phase or by spermidine depletion. The increased rate of eIF-5A modification in DH23A/b cells is due to an increased accumulation of the unmodified eIF-5A precursor. Increased precursor accumulation is not due to increased eIF-5A transcription, but rather it can be attributed to a metabolic accumulation caused by growth under conditions of chronically limiting spermidine. Selection using drugs that inhibit ODC apparently does not cause alterations in the eIF-5A modification pathway. These data support the hypothesis that one of the main effects of spermidine depletion is depletion of the modified eIF-5A pool, and that this is a critical factor in the cytostasis often observed after depletion of cellular polyamines.

  17. Defensins: antifungal lessons from eukaryotes

    Directory of Open Access Journals (Sweden)

    Patrícia M. Silva

    2014-03-01

    Full Text Available Over the last years, antimicrobial peptides (AMPs have been the focus of intense research towards the finding of a viable alternative to current antifungal drugs. Defensins are one of the major families of AMPs and the most represented among all eukaryotic groups, providing an important first line of host defense against pathogenic microorganisms. Several of these cysteine-stabilized peptides present a relevant effect against fungi. Defensins are the AMPs with the broader distribution across all eukaryotic kingdoms, namely, Fungi, Plantæ and Animalia, and were recently shown to have an ancestor in a bacterial organism. As a part of the host defense, defensins act as an important vehicle of information between innate and adaptive immune system and have a role in immunomodulation. This multidimensionality represents a powerful host shield, hard for microorganisms to overcome using single approach resistance strategies. Pathogenic fungi resistance to conventional antimycotic drugs is becoming a major problem. Defensins, as other AMPs, have shown to be an effective alternative to the current antimycotic therapies, demonstrating potential as novel therapeutic agents or drug leads. In this review, we summarize the current knowledge on some eukaryotic defensins with antifungal action. An overview of the main targets in the fungal cell and the mechanism of action of these AMPs (namely, the selectivity for some fungal membrane components are presented. Additionally, recent works on antifungal defensins structure, activity and citotoxicity are also reviewed.

  18. Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

    Science.gov (United States)

    Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

    2015-01-01

    DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

  19. Septins and the lateral compartmentalization of eukaryotic membranes.

    Science.gov (United States)

    Caudron, Fabrice; Barral, Yves

    2009-04-01

    Eukaryotic cells from neurons and epithelial cells to unicellular fungi frequently rely on cellular appendages such as axons, dendritic spines, cilia, and buds for their biology. The emergence and differentiation of these appendages depend on the formation of lateral diffusion barriers at their bases to insulate their membranes from the rest of the cell. Here, we review recent progress regarding the molecular mechanisms and functions of such barriers. This overview underlines the importance and conservation of septin-dependent diffusion barriers, which coordinately compartmentalize both plasmatic and internal membranes. We discuss their role in memory establishment and the control of cellular aging.

  20. Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase-Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase.

    Science.gov (United States)

    Sahu, Divya; Gupta, Sounak; Hau, Andrew M; Nakashima, Kazufumi; Leivo, Mariah Z; Searles, Stephen C; Elson, Paul; Bomalaski, John S; Casteel, Darren E; Boss, Gerry R; Hansel, Donna E

    2016-12-09

    Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

  1. Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A mammalian expression plasmid pcDNA3.1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3.1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BamH Ⅰ and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot.Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3.1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3.1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.

  2. Expression of acyl-lipid Delta12-desaturase gene in prokaryotic and eukaryotic cells and its effect on cold stress tolerance of potato.

    Science.gov (United States)

    Amiri, Reza Maali; Yur'eva, Natalia O; Shimshilashvili, Khristina R; Goldenkova-Pavlova, Irina V; Pchelkin, Vasiliy P; Kuznitsova, Elmira I; Tsydendambaev, Vladimir D; Trunova, Tamara I; Los, Dmitry A; Jouzani, Gholamreza Salehi; Nosov, Alexander M

    2010-03-01

    We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

  3. External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

    Science.gov (United States)

    Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

    2010-07-23

    Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

  4. [Construction of recombinant human nerve growth factor (rh-β-NGF) eukaryotic vector and its expression in HEK293 cells].

    Science.gov (United States)

    Li, Jingchuan; Xue, Bofu; Yuan, Yuan; Ma, Mo; Zhu, Lin; Milburn, Rebecca; Le, Li; Hu, Peizhen; Ye, Jing

    2015-03-01

    Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.

  5. Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR.

    Science.gov (United States)

    Carapuça, Elisabete; Azzoni, Adriano R; Prazeres, Duarte M F; Monteiro, Gabriel A; Mergulhão, Filipe J M

    2007-10-01

    A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 x 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

  6. Transient expression of a mitochondrial precursor protein - A new approach to study mitochondrial protein import in cells of higher eukaryotes

    NARCIS (Netherlands)

    Huckriede, A; Heikema, A; Wilschut, J; Agsteribbe, E

    1996-01-01

    In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly effici

  7. Comparative genomics of Eukaryotes

    NARCIS (Netherlands)

    Noort, Vera van

    2007-01-01

    This thesis focuses on developing comparative genomics methods in eukaryotes, with an emphasis on applications for gene function prediction and regulatory element detection. In the past, methods have been developed to predict functional associations between gene pairs in prokaryotes. The challenge

  8. Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

    Science.gov (United States)

    Risco, Cristina; Sanmartín-Conesa, Eva; Tzeng, Wen-Pin; Frey, Teryl K.; Seybold, Volker; de Groot, Raoul J.

    2012-01-01

    Summary More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell’s interior are lagging behind. We describe a novel approach, metal-tagging TEM (METTEM) that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before. PMID:22579245

  9. Specific histone modifications play critical roles in the control of encystation and antigenic variation in the early-branching eukaryote Giardia lamblia.

    Science.gov (United States)

    Carranza, Pedro G; Gargantini, Pablo R; Prucca, César G; Torri, Alessandro; Saura, Alicia; Svärd, Staffan; Lujan, Hugo D

    2016-12-01

    During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post-transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post-translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD(+)-dependent and NAD(+)-independent histone deacetylases regulate encystation; however, a unique NAD(+)-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Quantitative imaging of gold and silver nanoparticles in single eukaryotic cells by laser ablation ICP-MS.

    Science.gov (United States)

    Drescher, Daniela; Giesen, Charlotte; Traub, Heike; Panne, Ulrich; Kneipp, Janina; Jakubowski, Norbert

    2012-11-20

    Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.

  11. Precambrian Skeletonized Microbial Eukaryotes

    Science.gov (United States)

    Lipps, Jere H.

    2017-04-01

    Skeletal heterotrophic eukaryotes are mostly absent from the Precambrian, although algal eukaryotes appear about 2.2 billion years ago. Tintinnids, radiolaria and foraminifera have molecular origins well back into the Precambrian yet no representatives of these groups are known with certainty in that time. These data infer times of the last common ancestors, not the appearance of true representatives of these groups which may well have diversified or not been preserved since those splits. Previous reports of these groups in the Precambrian are misinterpretations of other objects in the fossil record. Reported tintinnids at 1600 mya from China are metamorphic shards or mineral artifacts, the many specimens from 635-715 mya in Mongolia may be eukaryotes but they are not tintinnids, and the putative tintinnids at 580 mya in the Doushantou formation of China are diagenetic alterations of well-known acritarchs. The oldest supposed foraminiferan is Titanotheca from 550 to 565 mya rocks in South America and Africa is based on the occurrence of rutile in the tests and in a few modern agglutinated foraminifera, as well as the agglutinated tests. Neither of these nor the morphology are characteristic of foraminifera; hence these fossils remain as indeterminate microfossils. Platysolenites, an agglutinated tube identical to the modern foraminiferan Bathysiphon, occurs in the latest Neoproterozoic in Russia, Canada, and the USA (California). Some of the larger fossils occurring in typical Ediacaran (late Neoproterozoic) assemblages may be xenophyophorids (very large foraminifera), but the comparison is disputed and flawed. Radiolaria, on occasion, have been reported in the Precambrian, but the earliest known clearly identifiable ones are in the Cambrian. The only certain Precambrian heterotrophic skeletal eukaryotes (thecamoebians) occur in fresh-water rocks at about 750 mya. Skeletonized radiolaria and foraminifera appear sparsely in the Cambrian and radiate in the Ordovician

  12. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part C: Protein Synthesis and Post-Translational Processing in Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The translation of mRNA constitutes the first step in the synthesis of a functional protein. The polypeptide chain is subsequently folded into the appropriate three-dimensional configuration and undergoes a variety of processing steps before being converted into its active form. These processing steps are intimately related to the cellular events that occur in the endoplasmic reticulum and Golgi compartments, and determine the sorting and transport of different proteins to their appropriate destinations within the cell. While the regulation of gene expression occurs primarily at the level of transcription, the expression of many genes can also be controlled at the level of translation. Most proteins can be regulated in response to extracellular signals. In addition, intracellular protein levels can be controlled by differential rates of protein degradation. Thus, the regulation of both the amounts and activities of intracellular proteins ultimately determines all aspects of cell behaviour.

  13. Development of a soluble PTD-HPV18E7 fusion protein and its functional characterization in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Xiaofei Yan; Shah Walayat; Qinfeng Shi; Jin Zheng; Yili Wang

    2009-01-01

    Though accumulated evidence has demonstrated the transformation capacity of human papillomavirus (HPV) type 18 protein E7, the underlying mechanism is still arguable. Developing a protein transduction domain (PTD)-iinked E7 molecule is a suitable strategy for assessing the biological functions of the protein. In the present study, HPVI8 E7 protein fused to an N-terminal PTD was expressed in the form of giutathione S-trans-ferase fusion protein in Escherichia coil with pGEX-4T-3 vector. After giutathione-Sepharose 4B bead affinity purification, immunobiot identification and thrombin cleavage, the PTD-18E7 protein showed structural and functional activity in that it potently transduced the cells and localized into their nuclei. The PTD-18E7 protein transduced the NIH3T3 cells in 30 min and remained stable for at least 24 h. In addition, the PTD-18E7 protein interacted with retinoblastoma protein (pRB) and caused pRB degradation in the transduced NIH3T3 cells. In contrast to the pRB level, p27 protein level was elevated in the transduced NIH3T3 cells. The PTD-18E7 protein gives us a new tool to study the biological functions of the HPV E7 protein.

  14. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    NARCIS (Netherlands)

    Blecha, Andreas; Zarschler, Kristof; Sjollema, Klaas A.; Veenhuis, Marten; Rödel, Gerhard; Rodel, G.

    2005-01-01

    Background: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested

  15. The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

    Science.gov (United States)

    Mittal, Rohit; Peak-Chew, Sew Yeu; Sade, Robert S.; Vallis, Yvonne; McMahon, Harvey T.

    2010-01-01

    Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP6). We extend the applicability of our results to show that IP6 also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP6-induced conformational change in AvrA suggests that IP6 acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP6; once injected into mammalian cells by the pathogen these toxins bind host cell IP6, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity. PMID:20430892

  16. Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth▿

    OpenAIRE

    2007-01-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  17. [Expression of mutation type GJA8 gene and wild type GJA8 gene of a congenital inherited nuclear cataract family in eukaryotic cells].

    Science.gov (United States)

    Zheng, Jian-qiu; Liu, Ping; Wang, Jian-wen; Liu, Jian-ju

    2010-04-20

    To clone the sequence of mutation type GJA8 gene (mGJA8) and wild type GJA8 gene (wGJA8) of a congenital inherited nuclear cataract family and study their expression in eukaryotic cell lines in vitro. The mGJA8 and wGJA8 were amplified from this family's DNA and healthy people's DNA by PCR respectively. The mGJA8 and wGJA8 were recombined with plasmid pEGFP-N1 respectively. The accuracy of pEGFP-N1-GJA8 was confirmed by restriction enzyme digestion and DNA sequencing. Finally pEGFP-N1- mGJA8 and pEGFP-N1- wGJA8 and GFP protein were transfected into COS7 cells by lipofectin. The expression of pEGFP-N1-GJA8 and GFP fusion protein were to observe under fluorescence microscope, and to detect by Western-blotting and immunohistochemical staining. The mGJA8 and wGJA8 were cloned successfully. With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 were constructed correctly and their GFP fusions were expressed in transfected COS7 cells. The expression of pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 fusion protein were observed under fluorescence microscope, and detected by Western-blotting and immunohistochemical staining successfully. The mGJA8 gene and wGJA8 gene are cloned successfully, and pEGFP-N1-mGJA8 and pEGFP-N1-mGJA8 fusion protein can be expressed in COS7 cells, which establish the foundation for further studying the mechanism of this congenital inherited nuclear cataract family.

  18. A multifunctional region of the Shigella type 3 effector IpgB1 is important for secretion from bacteria and membrane targeting in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Sonia C P Costa

    Full Text Available Type 3 secretion systems are complex nanomachines used by many Gram-negative bacteria to deliver tens of proteins (effectors directly into host cells. Once delivered into host cells, effectors often target to specific cellular loci where they usurp host cell processes to their advantage. Here, using the yeast model system, we identify the membrane localization domain (MLD of IpgB1, a stretch of 20 amino acids enriched for hydrophobic residues essential for the targeting of this effector to the plasma membrane. Embedded within these residues are ten that define the IpgB1 chaperone-binding domain for Spa15. As observed with dedicated class IA chaperones that mask hydrophobic MLDs, Spa15, a class IB chaperone, promotes IpgB1 stability by binding this hydrophobic region. However, despite being stable, an IpgB1 allele that lacks the MLD is not recognized as a secreted substrate. Similarly, deletion of the chaperone binding domains of IpgB1 and three additional Spa15-dependent effectors result in alleles that are no longer recognized as secreted substrates despite the presence of intact N-terminal secretion signal sequences. This is in contrast with MLD-containing effectors that bind class IA dedicated chaperones, as deletion of the MLD of these effectors alleviates the chaperone requirement for secretion. These observations indicate that at least for substrates of class IB chaperones, the chaperone-effector complex plays a major role in defining type 3 secreted proteins and highlight how a single region of an effector can play important roles both within prokaryotic and eukaryotic cells.

  19. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  20. Cell-Size Control

    Science.gov (United States)

    Amodeo, Amanda A.; Skotheim, Jan M.

    2015-01-01

    Cells of a given type maintain a characteristic cell size to function efficiently in their ecological or organismal context. They achieve this through the regulation of growth rates or by actively sensing size and coupling this signal to cell division. We focus this review on potential size-sensing mechanisms, including geometric, external cue, and titration mechanisms. Mechanisms that titrate proteins against DNA are of particular interest because they are consistent with the robust correlation of DNA content and cell size. We review the literature, which suggests that titration mechanisms may underlie cell-size sensing in Xenopus embryos, budding yeast, and Escherichia coli, whereas alternative mechanisms may function in fission yeast. PMID:26254313

  1. In vitro Expression in Eukaryotic Cells of a Prion Protein Gene Cloned from Scrapie-Infected Mouse Brain

    Science.gov (United States)

    Caughey, Byron; Race, Richard E.; Vogel, Mari; Buchmeier, Michael J.; Chesebro, Bruce

    1988-07-01

    It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

  2. DNA polymerase zeta (polζ) in higher eukaryotes

    Institute of Scientific and Technical Information of China (English)

    Gregory N Gan; John P Wittschieben; Birgitte φ Wittschieben; Richard D Wood

    2008-01-01

    Most current knowledge about DNA polymerase zeta (pol ζ) comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where polζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Rev1. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already appar-ent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genuine instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7(MAD2B) in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair ofinterstrand DNA erosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent devel-opments in these areas.

  3. Controlling Cell Function with Geometry

    Science.gov (United States)

    Mrksich, Milan

    2012-02-01

    This presentation will describe the use of patterned substrates to control cell shape with examples that illustrate the ways in which cell shape can regulate cell function. Most cells are adherent and must attach to and spread on a surface in order to survive, proliferate and function. In tissue, this surface is the extracellular matrix (ECM), an insoluble scaffold formed by the assembly of several large proteins---including fibronectin, the laminins and collagens and others---but in the laboratory, the surface is prepared by adsorbing protein to glass slides. To pattern cells, gold-coated slides are patterned with microcontact printing to create geometric features that promote cell attachment and that are surrounded by inert regions. Cells attach to these substrates and spread to adopt the shape defined by the underlying pattern and remain stable in culture for several days. Examples will be described that used a series of shapes to reveal the relationship between the shape of the cell and the structure of its cytoskeleton. These geometric cues were used to control cell polarity and the tension, or contractility, present in the cytoskeleton. These rules were further used to control the shapes of mesenchymal stem cells and in turn to control the differentiation of these cells into specialized cell types. For example, stem cells that were patterned into a ``star'' shape preferentially differentiated into bone cells whereas those that were patterned into a ``flower'' shape preferred a fat cell fate. These influences of shape on differentiation depend on the mechanical properties of the cytoskeleton. These examples, and others, reveal that shape is an important cue that informs cell function and that can be combined with the more common soluble cues to direct and study cell function.

  4. In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types.

    Science.gov (United States)

    Sadiq, Rakhshinda; Khan, Qaiser Mahmood; Mobeen, Ameena; Hashmat, Amer Jamal

    2015-04-01

    Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

  5. Diagnostic tool based on an HTLV-1-Tax expression system in eukaryotic cells using a poxvirus vector.

    Science.gov (United States)

    de Souza, Jaqueline Gontijo; Fonseca, Flávio Guimarães da; Martins-Filho, Olindo Assis; Teixeira-Carvalho, Andrea; Martins, Camila Pacheco Silveira; Carvalho, Luciana Debortoli; Coelho-Dos-Reis, Jordana Grazziela Alves; Barbosa-Stancioli, Edel Figueiredo

    2010-06-01

    Human T-lymphotropic virus 1 (HTLV-1) induces an immune-mediated inflammatory disease affecting the nervous system that eventually is accompanied by ocular, rheumatic and dermatologic manifestations (HTLV-1 associated myelopathy/tropical spastic paraparesis, or HAM/TSP). Proviral load and HTLV-1 protein expression, mainly of Tax, is correlated with disease progression and induction of host-virus equilibrium breakdown that, reportedly, involves the presence of Tax-specific cytotoxic T lymphocytes (CTL), T regulatory cells and anti-Tax antibodies. Based on knowledge of anti-Tax antibodies as markers of disease progression, the objectives of this study were both to design an infection/transfection system using the Vaccinia virus and a tax-encoding plasmid for the expression of Tax protein as well as to use this cell support to evaluate anti-Tax IgG by flow cytometry. The flow cytometry assay was standardized using pooled sera from each test group (negative, asymptomatic and HAM/TSP patients). The HAM/TSP group presented higher IgG anti-Tax reactivity (above 70%) than the asymptomatic group (nearly 40% reactivity). The data indicate that the infection/transfection system is useful for assessing Tax expression. This is a promising assay for use as a diagnostic tool to detect IgG anti-Tax and monitor HTLV-1 infected individuals.

  6. Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

    Science.gov (United States)

    Stratikos, E.; Alberdi, E.; Gettins, P. G.; Becerra, S. P.

    1996-01-01

    Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation. PMID:8976566

  7. Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang

    2005-01-01

    Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

  8. Atypical mitochondrial inheritance patterns in eukaryotes.

    Science.gov (United States)

    Breton, Sophie; Stewart, Donald T

    2015-10-01

    Mitochondrial DNA (mtDNA) is predominantly maternally inherited in eukaryotes. Diverse molecular mechanisms underlying the phenomenon of strict maternal inheritance (SMI) of mtDNA have been described, but the evolutionary forces responsible for its predominance in eukaryotes remain to be elucidated. Exceptions to SMI have been reported in diverse eukaryotic taxa, leading to the prediction that several distinct molecular mechanisms controlling mtDNA transmission are present among the eukaryotes. We propose that these mechanisms will be better understood by studying the deviations from the predominating pattern of SMI. This minireview summarizes studies on eukaryote species with unusual or rare mitochondrial inheritance patterns, i.e., other than the predominant SMI pattern, such as maternal inheritance of stable heteroplasmy, paternal leakage of mtDNA, biparental and strictly paternal inheritance, and doubly uniparental inheritance of mtDNA. The potential genes and mechanisms involved in controlling mitochondrial inheritance in these organisms are discussed. The linkage between mitochondrial inheritance and sex determination is also discussed, given that the atypical systems of mtDNA inheritance examined in this minireview are frequently found in organisms with uncommon sexual systems such as gynodioecy, monoecy, or andromonoecy. The potential of deviations from SMI for facilitating a better understanding of a number of fundamental questions in biology, such as the evolution of mtDNA inheritance, the coevolution of nuclear and mitochondrial genomes, and, perhaps, the role of mitochondria in sex determination, is considerable.

  9. Evolution of cell cycle control: same molecular machines, different regulation

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Brunak, Søren

    2007-01-01

    layers of regulation together control the activity of cell cycle complexes and how this regulation has evolved. The results show surprisingly poor conservation of both the transcriptional and the post-translation regulation of individual genes and proteins; however, the changes in one layer of regulation...... or deactivated at specific stages during the cell cycle through a wide variety of mechanisms including transcriptional regulation, phosphorylation, subcellular translocation and targeted degradation. In a series of integrative analyses of different genome-scale data sets, we have studied how these different......Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated...

  10. The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

    Energy Technology Data Exchange (ETDEWEB)

    Fritz-Laylin, Lillian K.; Prochnik, Simon E.; Ginger, Michael L.; Dacks, Joel; Carpenter, Meredith L.; Field, Mark C.; Kuo, Alan; Paredez, Alex; Chapman, Jarrod; Pham, Jonathan; Shu, Shengqiang; Neupane, Rochak; Cipriano, Michael; Mancuso, Joel; Tu, Hank; Salamov, Asaf; Lindquist, Erika; Shapiro, Harris; Lucas, Susan; Grigoriev, Igor V.; Cande, W. Zacheus; Fulton, Chandler; Rokhsar, Daniel S.; Dawson, Scott C.

    2010-03-01

    Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

  11. Senescence marker protein 30 (SMP30 expression in eukaryotic cells: existence of multiple species and membrane localization.

    Directory of Open Access Journals (Sweden)

    Peethambaran Arun

    Full Text Available Senescence marker protein (SMP30, also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+ homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA. Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ∼28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of β and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.

  12. Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium.

    Science.gov (United States)

    Wiejak, Jolanta; Surmacz, Liliana; Wyroba, Elzbieta

    2004-04-01

    Evidence that dynamin is associated with the sequestration of the Paramecium beta(2)-adrenergic receptor (betaAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of betaAR analogue in the subcellular fractions upon isoproterenol treatment: it is redistributed from the membraneous to the cytosolic fraction, as revealed by quantitative image analysis of western blots. Here we confirm and extend this observation by laser scanning confocal and immunogold electron microscopy. In the presence of isoproterenol (10 micro mol l(-1)) betaAR translocated from the cell surface into dynamin-positive vesicles in the cytoplasmic compartment, as observed by dual fluorochrome immunolabeling in a series of the confocal optical sections. Colocalization of betaAR and dynamin in the tiny endocytic vesicles was detected by further electron microscopic studies. Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to the beta-adrenergic receptor kinase (betaARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human betaARK2 (GRK3), and may participate in Paramecium betaAR desensitization. These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate betaAR exists in unicellular PARAMECIUM:

  13. Eukaryotic membrane protein overproduction in Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Chan, Ka Wai; Slotboom, Dirk Jan; Floyd, Suzanne; O’Connor, Rosemary; Monné, Magnus

    2005-01-01

    Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has

  14. Energetics and genetics across the prokaryote-eukaryote divide

    Directory of Open Access Journals (Sweden)

    Lane Nick

    2011-06-01

    Full Text Available Abstract Background All complex life on Earth is eukaryotic. All eukaryotic cells share a common ancestor that arose just once in four billion years of evolution. Prokaryotes show no tendency to evolve greater morphological complexity, despite their metabolic virtuosity. Here I argue that the eukaryotic cell originated in a unique prokaryotic endosymbiosis, a singular event that transformed the selection pressures acting on both host and endosymbiont. Results The reductive evolution and specialisation of endosymbionts to mitochondria resulted in an extreme genomic asymmetry, in which the residual mitochondrial genomes enabled the expansion of bioenergetic membranes over several orders of magnitude, overcoming the energetic constraints on prokaryotic genome size, and permitting the host cell genome to expand (in principle over 200,000-fold. This energetic transformation was permissive, not prescriptive; I suggest that the actual increase in early eukaryotic genome size was driven by a heavy early bombardment of genes and introns from the endosymbiont to the host cell, producing a high mutation rate. Unlike prokaryotes, with lower mutation rates and heavy selection pressure to lose genes, early eukaryotes without genome-size limitations could mask mutations by cell fusion and genome duplication, as in allopolyploidy, giving rise to a proto-sexual cell cycle. The side effect was that a large number of shared eukaryotic basal traits accumulated in the same population, a sexual eukaryotic common ancestor, radically different to any known prokaryote. Conclusions The combination of massive bioenergetic expansion, release from genome-size constraints, and high mutation rate favoured a protosexual cell cycle and the accumulation of eukaryotic traits. These factors explain the unique origin of eukaryotes, the absence of true evolutionary intermediates, and the evolution of sex in eukaryotes but not prokaryotes. Reviewers This article was reviewed by

  15. Universal Temporal Profile of Replication Origin Activation in Eukaryotes

    Science.gov (United States)

    Goldar, Arach

    2011-03-01

    The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

  16. Origin of phagotrophic eukaryotes as social cheaters in microbial biofilms

    Directory of Open Access Journals (Sweden)

    Jékely Gáspár

    2007-01-01

    Full Text Available Abstract Background The origin of eukaryotic cells was one of the most dramatic evolutionary transitions in the history of life. It is generally assumed that eukaryotes evolved later then prokaryotes by the transformation or fusion of prokaryotic lineages. However, as yet there is no consensus regarding the nature of the prokaryotic group(s ancestral to eukaryotes. Regardless of this, a hardly debatable fundamental novel characteristic of the last eukaryotic common ancestor was the ability to exploit prokaryotic biomass by the ingestion of entire cells, i.e. phagocytosis. The recent advances in our understanding of the social life of prokaryotes may help to explain the origin of this form of total exploitation. Presentation of the hypothesis Here I propose that eukaryotic cells originated in a social environment, a differentiated microbial mat or biofilm that was maintained by the cooperative action of its members. Cooperation was costly (e.g. the production of developmental signals or an extracellular matrix but yielded benefits that increased the overall fitness of the social group. I propose that eukaryotes originated as selfish cheaters that enjoyed the benefits of social aggregation but did not contribute to it themselves. The cheaters later evolved into predators that lysed other cells and eventually became professional phagotrophs. During several cycles of social aggregation and dispersal the number of cheaters was contained by a chicken game situation, i.e. reproductive success of cheaters was high when they were in low abundance but was reduced when they were over-represented. Radical changes in cell structure, including the loss of the rigid prokaryotic cell wall and the development of endomembranes, allowed the protoeukaryotes to avoid cheater control and to exploit nutrients more efficiently. Cellular changes were buffered by both the social benefits and the protective physico-chemical milieu of the interior of biofilms. Symbiosis

  17. How eukaryotic genes are transcribed.

    Science.gov (United States)

    Venters, Bryan J; Pugh, B Franklin

    2009-06-01

    Regulation of eukaryotic gene expression is far more complex than one might have imagined 30 years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: (1) a well-defined organization of nucleosomes and modification states at most genes; (2) regulatory networks of sequence-specific transcription factors; (3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II; and (4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation.

  18. Cell Control Engineering

    DEFF Research Database (Denmark)

    Lynggaard, Hans Jørgen Birk; Alting, Leo

    1996-01-01

    strategy. The concept has been validated and verified at Odense Steel Shipyard Ltd. in several robotic arc welding systems in the production using an application enabler, UNIX, X, SQL and (wireless) Ethernet. Integration with off-line programming, production planning, monitoring and maintenance has been...... of any production installation and in addition the software element is often very critical as it is the integrating, controlling and co-ordinating system component. As such, a CCE concept providing high quality and high functionality as well as flexibility is an important aspect of a company's automation...

  19. Genome-wide analysis of the phosphoinositide kinome from two ciliates reveals novel evolutionary links for phosphoinositide kinases in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    George Leondaritis

    Full Text Available BACKGROUND: The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. CONCLUSIONS/SIGNIFICANCE: Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory

  20. On eukaryotic intelligence: signaling system's guidance in the evolution of multicellular organization.

    Science.gov (United States)

    Marijuán, Pedro C; del Moral, Raquel; Navarro, Jorge

    2013-10-01

    Communication with the environment is an essential characteristic of the living cell, even more when considering the origins and evolution of multicellularity. A number of changes and tinkering inventions were necessary in the evolutionary transition between prokaryotic and eukaryotic cells, which finally made possible the appearance of genuine multicellular organisms. In the study of this process, however, the transformations experimented by signaling systems themselves have been rarely object of analysis, obscured by other more conspicuous biological traits: incorporation of mitochondria, segregated nucleus, introns/exons, flagellum, membrane systems, etc. Herein a discussion of the main avenues of change from prokaryotic to eukaryotic signaling systems and a review of the signaling resources and strategies underlying multicellularity will be attempted. In the expansion of prokaryotic signaling systems, four main systemic resources were incorporated: molecular tools for detection of solutes, molecular tools for detection of solvent (Donnan effect), the apparatuses of cell-cycle control, and the combined system endocytosis/cytoskeleton. The multiple kinds of enlarged, mixed pathways that emerged made possible the eukaryotic revolution in morphological and physiological complexity. The massive incorporation of processing resources of electro-molecular nature, derived from the osmotic tools counteracting the Donnan effect, made also possible the organization of a computational tissue with huge information processing capabilities: the nervous system. In the central nervous systems of vertebrates, and particularly in humans, neurons have achieved both the highest level of molecular-signaling complexity and the highest degree of information-processing adaptability. Theoretically, it can be argued that there has been an accelerated pace of evolutionary change in eukaryotic signaling systems, beyond the other general novelties introduced by eukaryotic cells in their

  1. More Comprehensive Understanding of the Differences Between Prokaryotic Cells and Eukaryotic Cells%应较为全面地理解原核细胞与真核细胞的差异

    Institute of Scientific and Technical Information of China (English)

    赵晓平

    2015-01-01

    原核细胞(prokaryotic cell)与真核细胞(eukaryotic cell)的进化水平不相同,在结构组成、生态功能、基因组特点、蛋白质翻译、基因的表达与调控等多方面存在着明显的差异和区别,较为全面地了解这些差异和区别,对于学好和掌握细胞生物学、遗传学、分子生物学等课程的相关内容会有所帮助和启发.

  2. An evolutionary network of genes present in the eukaryote common ancestor polls genomes on eukaryotic and mitochondrial origin.

    Science.gov (United States)

    Thiergart, Thorsten; Landan, Giddy; Schenk, Marc; Dagan, Tal; Martin, William F

    2012-01-01

    To test the predictions of competing and mutually exclusive hypotheses for the origin of eukaryotes, we identified from a sample of 27 sequenced eukaryotic and 994 sequenced prokaryotic genomes 571 genes that were present in the eukaryote common ancestor and that have homologues among eubacterial and archaebacterial genomes. Maximum-likelihood trees identified the prokaryotic genomes that most frequently contained genes branching as the sister to the eukaryotic nuclear homologues. Among the archaebacteria, euryarchaeote genomes most frequently harbored the sister to the eukaryotic nuclear gene, whereas among eubacteria, the α-proteobacteria were most frequently represented within the sister group. Only 3 genes out of 571 gave a 3-domain tree. Homologues from α-proteobacterial genomes that branched as the sister to nuclear genes were found more frequently in genomes of facultatively anaerobic members of the rhiozobiales and rhodospirilliales than in obligate intracellular ricketttsial parasites. Following α-proteobacteria, the most frequent eubacterial sister lineages were γ-proteobacteria, δ-proteobacteria, and firmicutes, which were also the prokaryote genomes least frequently found as monophyletic groups in our trees. Although all 22 higher prokaryotic taxa sampled (crenarchaeotes, γ-proteobacteria, spirochaetes, chlamydias, etc.) harbor genes that branch as the sister to homologues present in the eukaryotic common ancestor, that is not evidence of 22 different prokaryotic cells participating at eukaryote origins because prokaryotic "lineages" have laterally acquired genes for more than 1.5 billion years since eukaryote origins. The data underscore the archaebacterial (host) nature of the eukaryotic informational genes and the eubacterial (mitochondrial) nature of eukaryotic energy metabolism. The network linking genes of the eukaryote ancestor to contemporary homologues distributed across prokaryotic genomes elucidates eukaryote gene origins in a dialect

  3. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  4. Cell biology. Metabolic control of cell death.

    Science.gov (United States)

    Green, Douglas R; Galluzzi, Lorenzo; Kroemer, Guido

    2014-09-19

    Beyond their contribution to basic metabolism, the major cellular organelles, in particular mitochondria, can determine whether cells respond to stress in an adaptive or suicidal manner. Thus, mitochondria can continuously adapt their shape to changing bioenergetic demands as they are subjected to quality control by autophagy, or they can undergo a lethal permeabilization process that initiates apoptosis. Along similar lines, multiple proteins involved in metabolic circuitries, including oxidative phosphorylation and transport of metabolites across membranes, may participate in the regulated or catastrophic dismantling of organelles. Many factors that were initially characterized as cell death regulators are now known to physically or functionally interact with metabolic enzymes. Thus, several metabolic cues regulate the propensity of cells to activate self-destructive programs, in part by acting on nutrient sensors. This suggests the existence of "metabolic checkpoints" that dictate cell fate in response to metabolic fluctuations. Here, we discuss recent insights into the intersection between metabolism and cell death regulation that have major implications for the comprehension and manipulation of unwarranted cell loss.

  5. Eukaryotic transcription factors

    DEFF Research Database (Denmark)

    Staby, Lasse; O'Shea, Charlotte; Willemoës, Martin

    2017-01-01

    Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains...... targeting specific cis-acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID...... them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions...

  6. Effects of xenobiotic compounds on the cell activities of Euplotes crassus, a single-cell eukaryotic test organism for the study of the pollution of marine sediments

    Energy Technology Data Exchange (ETDEWEB)

    Trielli, Francesca [Dipartimento per lo Studio del Territorio e delle sue Risorse, University of Genoa Corso Europa, 26, I-16132 Genova (Italy); Amaroli, Andrea [Dipartimento per lo Studio del Territorio e delle sue Risorse, University of Genoa Corso Europa, 26, I-16132 Genova (Italy); Sifredi, Francesca [Dipartimento per lo Studio del Territorio e delle sue Risorse, University of Genoa Corso Europa, 26, I-16132 Genova (Italy); Marchi, Barbara [Dipartimento di Biologia, University of Genoa, Viale Benedetto XV, 5, I-16132 Genova (Italy); Falugi, Carla [Dipartimento di Biologia, University of Genoa, Viale Benedetto XV, 5, I-16132 Genova (Italy); Corrado, Maria Umberta Delmonte [Dipartimento per lo Studio del Territorio e delle sue Risorse, University of Genoa Corso Europa, 26, I-16132 Genova (Italy)]. E-mail: corrado@dipteris.unige.it

    2007-08-01

    It is now widely accepted that assays with protists are relevant to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of estuarine and coastal sediments was evaluated. For this purpose, the effects of exposure to pollutants were tested on the cell viability, fission rate and lysosomal membrane stability of E. crassus. The following toxicants were used: an organophosphate (OP) pesticide, basudin, an organochlorine hydrocarbon, AFD25, both employed especially for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl{sub 2}) and different mixtures of the above-mentioned compounds, as they might occur in polluted sites. Exposure to these toxicants affected cell viability at concentrations ranging from 96.6 to 966 x 10{sup 3} mg/l for basudin, from 3.3 to 33 x 10{sup 3} mg/l for AFD25 and from 0.1 to 1 mg/l for HgCl{sub 2}. A significant decrease in the mean fission rate (P < 0.001) was found after 24- or 48-h exposures to 9.66 mg/l basudin, 3.3 mg/l AFD25 and 7 x 10{sup -2} mg/l HgCl{sub 2}. Furthermore, the Neutral Red Retention Assay showed a significant decrease in lysosomal membrane stability after 60- and 120-min exposures to AFD25 (33 mg/l) and HgCl{sub 2} (0.33 mg/l). In addition, as it is well-known that the inhibition of acetylcholinesterase activity represents a specific biomarker of exposure to OP and carbamate pesticides in higher organisms, initially the presence of cholinesterase (ChE) activity was detected in E. crassus, using cytochemical, spectrophotometric and electrophoretic methods. Afterwards, this enzyme activity was characterized spectrophotometrically by its sensitivity to specific ChE inhibitors and to variations in pH and temperature. The ChE activity was inhibited significantly by basudin- (9.66 and 96.6 mg/l) or AFD25-exposure (3.3 mg/l). Conversely

  7. Molecular paleontology and complexity in the last eukaryotic common ancestor.

    Science.gov (United States)

    Koumandou, V Lila; Wickstead, Bill; Ginger, Michael L; van der Giezen, Mark; Dacks, Joel B; Field, Mark C

    2013-01-01

    Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.

  8. Molecular paleontology and complexity in the last eukaryotic common ancestor

    Science.gov (United States)

    Koumandou, V. Lila; Wickstead, Bill; Ginger, Michael L.; van der Giezen, Mark; Dacks, Joel B.

    2013-01-01

    Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself. PMID:23895660

  9. Development of a radiation track structure clustering algorithm for the prediction of DNA DSB yields and radiation induced cell death in Eukaryotic cells

    Science.gov (United States)

    Douglass, Michael; Bezak, Eva; Penfold, Scott

    2015-04-01

    The preliminary framework of a combined radiobiological model is developed and calibrated in the current work. The model simulates the production of individual cells forming a tumour, the spatial distribution of individual ionization events (using Geant4-DNA) and the stochastic biochemical repair of DNA double strand breaks (DSBs) leading to the prediction of survival or death of individual cells. In the current work, we expand upon a previously developed tumour generation and irradiation model to include a stochastic ionization damage clustering and DNA lesion repair model. The Geant4 code enabled the positions of each ionization event in the cells to be simulated and recorded for analysis. An algorithm was developed to cluster the ionization events in each cell into simple and complex double strand breaks. The two lesion kinetic (TLK) model was then adapted to predict DSB repair kinetics and the resultant cell survival curve. The parameters in the cell survival model were then calibrated using experimental cell survival data of V79 cells after low energy proton irradiation. A monolayer of V79 cells was simulated using the tumour generation code developed previously. The cells were then irradiated by protons with mean energies of 0.76 MeV and 1.9 MeV using a customized version of Geant4. By replicating the experimental parameters of a low energy proton irradiation experiment and calibrating the model with two sets of data, the model is now capable of predicting V79 cell survival after low energy (<2 MeV) proton irradiation for a custom set of input parameters. The novelty of this model is the realistic cellular geometry which can be irradiated using Geant4-DNA and the method in which the double strand breaks are predicted from clustering the spatial distribution of ionisation events. Unlike the original TLK model which calculates a tumour average cell survival probability, the cell survival probability is calculated for each cell in the geometric tumour model

  10. A general strategy to construct small molecule biosensors in eukaryotes.

    Science.gov (United States)

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-12-29

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

  11. The control and execution of programmed cell death

    Energy Technology Data Exchange (ETDEWEB)

    Begum, R.; Pathak, N.; Hasnain, S.E.; Sah, N.K. [National Inst. of Immunology, New Delhi (India). Eukaryotic Gene Expression Lab.; Taneja, T.K.; Mohan, M. [National Inst. of Immunology, New Delhi (India). Eukaryotic Gene Expression Lab.]|[Dept. of Medical Elementology and Toxicology, New Delhi (India); Athar, M. [Dept. of Medical Elementology and Toxicology, New Delhi (India)

    1999-07-01

    Apoptosis or programmed cell death is a highly conserved genetically controlled response of metazoan cells to commit suicide. Non apoptotic programmed cell death seems to operate in single celled eukaryotes implying that evolution of PCD has preceded the evolution of multicellularity. PCD plays a crucial role in the regulation of cellular and tissue homeostasis and any aberrations in apoptosis leads to several diseases including cancer, neurodegenerative disorders and AIDS. The mechanisms by which apoptosis is controlled are varied. In some cells, members of bcl-2 family or p53 are crucial for regulating the apoptosis programme, whereas in other cells Fas ligand is more important. bcl-2 family members have a prime role in the regulation of cell death at all stages including development, whereas cell death during development is independent of p53. bcl-2 family members being localized on the outer mitochondrial membrane, control the mitochondrial homeostasis and cytochrome c redistribution and thereby regulate the cell death process. p53 promotes DNA damage mediated cell death after growth arrest and failed DNA repair. Caspases play a key role in the execution of cell death by mediating highly specific cleavages of crucial cellular proteins collectivley manifesting the apoptotic phenotype. Protein inhibitors like crm A, p35 and IAPs could prevent/control apoptosis induced by a broad array of cell death stimuli by several mechanisms specially interfering in caspase activation or caspase activity. Among endonucleases, caspase activated DNase (CAD) plays a crucial role in DNA fragmentation, a biochemical hallmark of apoptosis. As regulation of cell death seems to be as complex as regulation of cell proliferation, multiple kinase mediated regulatory mechanisms might control the apoptotic process. Thus, in spite of intensive research over the past few years, the field of apoptosis still remains fertile to unravel among others, the molecular mechanisms of cytochrome c

  12. Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells.

    OpenAIRE

    von Heijne, G

    1984-01-01

    A statistical analysis of the distribution of charged residues in the N-terminal region of 39 prokaryotic and 134 eukaryotic signal sequences reveals a remarkable similarity between the two samples, both in terms of net charge and in terms of the position of charged residues within the N-terminal region, and suggests that the formyl group on Metf is not removed in prokaryotic signal sequences.

  13. Eukaryotic diversity at pH extremes.

    Science.gov (United States)

    Amaral-Zettler, Linda A

    2012-01-01

    Extremely acidic (pH 9) environments support a diversity of single-cell and to a lesser extent, multicellular eukaryotic life. This study compared alpha and beta diversity in eukaryotic communities from seven diverse aquatic environments with pH values ranging from 2 to 11 using massively-parallel pyrotag sequencing targeting the V9 hypervariable region of the 18S ribosomal RNA (rRNA) gene. A total of 946 operational taxonomic units (OTUs) were recovered at a 6% cut-off level (94% similarity) across the sampled environments. Hierarchical clustering of the samples segregated the communities into acidic and alkaline groups. Similarity percentage (SIMPER) analysis followed by indicator OTU analysis (IOA) and non-metric multidimensional scaling (NMDS) were used to determine which characteristic groups of eukaryotic taxa typify acidic or alkaline extremes and the extent to which pH explains eukaryotic community structure in these environments. Spain's Rio Tinto yielded the fewest observed OTUs while Nebraska Sandhills alkaline lakes yielded the most. Distinct OTUs, including metazoan OTUs, numerically dominated pH extreme sites. Indicator OTUs included the diatom Pinnularia and unidentified opisthokonts (Fungi and Filasterea) in the extremely acidic environments, and the ciliate Frontonia across the extremely alkaline sites. Inferred from NMDS, pH explained only a modest fraction of the variation across the datasets, indicating that other factors influence the underlying community structure in these environments. The findings from this study suggest that the ability for eukaryotes to adapt to pH extremes over a broad range of values may be rare, but further study of taxa that can broadly adapt across diverse acidic and alkaline environments, respectively present good models for understanding adaptation and should be targeted for future investigations.

  14. Eukaryotic diversity at pH extremes

    Directory of Open Access Journals (Sweden)

    Linda A. Amaral-Zettler

    2013-01-01

    Full Text Available Extremely acidic (pH<3 and extremely alkaline (pH>9 environments support a diversity of single-cell and to a lesser extent, multicellular eukaryotic life. This study compared alpha and beta diversity in eukaryotic communities from 7 diverse aquatic environments with pH values ranging from 2 to 11 using massively-parallel pyrotag sequencing targeting the V9 hypervariable region of the 18S ribosomal RNA (rRNA gene. A total of 946 Operational Taxonomic Units (OTUs were recovered at a 6% cut-off level (94% similarity across the sampled environments. Hierarchical clustering of the samples segregated the communities into acidic and alkaline groups. Similarity Percentage Analysis (SIMPER followed by Indicator OTU Analysis (IOA and Non-metric Multidimensional Scaling (NMDS were used to determine which characteristic groups of eukaryotic taxa typify acidic or alkaline extremes and the extent to which pH explains eukaryotic community structure in these environments. Spain’s Rio Tinto yielded the fewest observed OTUs while Nebraska Sandhills alkaline lakes yielded the most. Distinct OTUs, including metazoan OTUs, numerically dominated pH extreme sites. Indicator OTUs included the diatom Pinnularia and unidentified opisthokonts (Fungi and Filasterea in the extremely acidic environments, and the ciliate Frontonia across the extremely alkaline sites. Inferred from NMDS, pH explained only a modest fraction of the variation across the datasets, indicating that other factors influence the underlying community structure in these environments. The findings from this study suggest that the ability for eukaryotes to adapt to pH extremes over a broad range of values may be rare, but further study of taxa that can broadly adapt across diverse acidic and alkaline environments respectively present good models for understanding adaptation and should be targeted for future investigations.

  15. Membranes, energetics, and evolution across the prokaryote-eukaryote divide

    Science.gov (United States)

    Lynch, Michael; Marinov, Georgi K

    2017-01-01

    The evolution of the eukaryotic cell marked a profound moment in Earth’s history, with most of the visible biota coming to rely on intracellular membrane-bound organelles. It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells. However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes. Thus, although the origin of the mitochondrion was a key event in evolutionary history, there is no reason to think membrane bioenergetics played a direct, causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity. DOI: http://dx.doi.org/10.7554/eLife.20437.001 PMID:28300533

  16. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard

    2015-02-01

    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  17. Arabinogalactan proteins have deep roots in eukaryotes

    DEFF Research Database (Denmark)

    Hervé, Cécile; Siméon, Amandine; Jam, Murielle

    2016-01-01

    is unrelated to land plants and green algae (Chloroplastida). Brown algae share common evolutionary features with other multicellular organisms, including a carbohydrate-rich cell wall. They differ markedly from plants in their cell wall composition, and AGPs have not been reported in brown algae. Here we......Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which...

  18. Expression of eukaryotic polypeptides in chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P

    2013-06-04

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  19. A genomic timescale for the origin of eukaryotes

    Directory of Open Access Journals (Sweden)

    Thompson Amanda S

    2001-09-01

    Full Text Available Abstract Background Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes. Results Eukaryotes were found to evolve faster than prokaryotes, with those eukaryotes derived from eubacteria evolving faster than those derived from archaebacteria. We found an early time of divergence (~4 billion years ago, Ga for archaebacteria and the archaebacterial genes in eukaryotes. Our analyses support at least two horizontal gene transfer events in the origin of eukaryotes, at 2.7 Ga and 1.8 Ga. Time estimates for the origin of cyanobacteria (2.6 Ga and the divergence of an early-branching eukaryote that lacks mitochondria (Giardia (2.2 Ga fall between those two events. Conclusions We find support for two symbiotic events in the origin of eukaryotes: one premitochondrial and a later mitochondrial event. The appearance of cyanobacteria immediately prior to the earliest undisputed evidence for the presence of oxygen (2.4–2.2 Ga suggests that the innovation of oxygenic photosynthesis had a relatively rapid impact on the environment as it set the stage for further evolution of the eukaryotic cell.

  20. Evaluation of the Toxicity of 5-Aryl-2-Aminoimidazole-Based Biofilm Inhibitors against Eukaryotic Cell Lines, Bone Cells and the Nematode Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Hans Steenackers

    2014-10-01

    Full Text Available Previously, we have synthesized several series of compounds based on the 5-aryl-2-aminoimidazole scaffold, which showed a preventive activity against microbial biofilms. We here studied the cytotoxicity of the most active compounds of each series. First, the cytostatic activity was investigated against a number of tumor cell lines (L1210, CEM and HeLa. A subset of monosubstituted 5-aryl-2-aminoimidazoles showed a moderate safety window, with therapeutic indices (TIs ranging between 3 and 20. Whereas introduction of a (cyclo-alkyl chain at the N1-position strongly reduced the TI, introduction of a (cyclo-alkyl chain or a triazole moiety at the 2N-position increased the TI up to 370. Since a promising application of preventive anti-biofilm agents is their use in anti-biofilm coatings for orthopedic implants, their effects on cell viability and functional behavior of human osteoblasts and bone marrow derived mesenchymal stem cells were tested. The 2N-substituted 5-aryl-2-aminoimidazoles consistently showed the lowest toxicity and allowed survival of the bone cells for up to 4 weeks. Moreover they did not negatively affect the osteogenic differentiation potential of the bone cells. Finally, we examined the effect of the compounds on the survival of Caenorhabditis elegans, which confirmed the higher safety window of 2N-substituted 5-aryl-2-aminoimidazoles.

  1. Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 齐义鹏; 朱朝晖; 鲁功成

    2002-01-01

    Summary: To explore a novel strategy for antisense gene therapy of cancer, the coding sequence ofhuman proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryoticvector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with li-posome. The PCNA expression in transferred cells was dynamically detected by immunofluo-rescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayedby MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryoticvector was successfully constructed and named as pLAPSN. After transfection with it for 1-7days, PCNA protein and mRNA levels in cancer cells were blocked by 16. 74 % - 84.21% (P<0. 05) and 23.27 % - 86.15 % (P<0. 05) respectively. The proliferation activities of transferredcells were inhibited by 27.91% - 62.07 % (P<0. 01), with cloning formation abilities being de-creased by 50. 81% (P<0. 01). It was concluded that the in vitro proliferation activities of cancercells could be effectively inhibited by blocking PCNA expression with antisense technique, whichcould serve as an ideal strategy for gene therapy of bladder cancer.

  2. Specific features of protein biosynthesis in higher eukaryotes

    Directory of Open Access Journals (Sweden)

    El’skaya A. V.

    2013-05-01

    Full Text Available Over 40 years of studies in the field of higher eukaryotic translation are summarized in the review. Among the pioneer results obtained we should especially accentuate the following: i discovery of the adaptation of tRNAs and aminoacyl-tRNA synthetases (ARSs cellular pools to the synthesis of specific proteins and modulation of the elongation rate by rare isoacceptor tRNAs; ii the chaperone-like properties of the translation components (ribosomes and elongation factor eEF1A; characterization of high molecular weight complexes of ARSs; iii functional compartmentalization, including channeling of tRNA in eukaryotic cells; iv molecular mechanisms of channeling mediated by different non-canonical complexes involving eEF1A, tRNA and aminoacyl-tRNA synthetases; v characterization of the crystal structure of eEF1A2; vi comparison of spatial structure, molecular dynamics, tyrosine phosphorylation and abilities to interact with different protein partners of the eEF1A1 and eEF1A2 isoforms; vii discovery of the microRNA-mediated control of the expression of the proto-oncogenic eEF1A2 isoform in cancer cells; viii examination of the cancer-related changes in translation elongation complex eEF1H and mechanisms of oncogene PTI-1 action; ix discovery of the third tRNA binding site on mammals ribosomes and the allosteric interaction of the 80S ribosomal A and E sites.

  3. A microbial avenue to cell cycle control in the plant superkingdom.

    Science.gov (United States)

    Tulin, Frej; Cross, Frederick R

    2014-10-01

    Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.

  4. Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes.

    Science.gov (United States)

    Gordon, E D; Mora, R; Meredith, S C; Lee, C; Lindquist, S L

    1987-12-05

    When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.

  5. Did group II intron proliferation in an endosymbiont-bearing archaeon create eukaryotes?

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2006-12-01

    Full Text Available Abstract Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the

  6. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

    Science.gov (United States)

    Wijffels, René H; Kruse, Olaf; Hellingwerf, Klaas J

    2013-06-01

    Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments. Cyanobacteria are promising host organisms for the production of small molecules that can be secreted such as ethanol, butanol, fatty acids and other organic acids. Eukaryotic microalgae are interesting for products for which cellular storage is important such as proteins, lipids, starch and alkanes. For the development of new and promising lines of production, strains of both cyanobacteria and eukaryotic microalgae have to be improved. Transformation systems have been much better developed in cyanobacteria. However, several products would be preferably produced with eukaryotic microalgae. In the case of cyanobacteria a synthetic-systems biology approach has a great potential to exploit cyanobacteria as cell factories. For eukaryotic microalgae transformation systems need to be further developed. A promising strategy is transformation of heterologous (prokaryotic and eukaryotic) genes in established eukaryotic hosts such as Chlamydomonas reinhardtii. Experimental outdoor pilots under containment for the production of genetically modified cyanobacteria and microalgae are in progress. For full scale production risks of release of genetically modified organisms need to be assessed. Copyright © 2013. Published by Elsevier Ltd.

  7. A statistical anomaly indicates symbiotic origins of eukaryotic membranes.

    Science.gov (United States)

    Bansal, Suneyna; Mittal, Aditya

    2015-04-01

    Compositional analyses of nucleic acids and proteins have shed light on possible origins of living cells. In this work, rigorous compositional analyses of ∼5000 plasma membrane lipid constituents of 273 species in the three life domains (archaea, eubacteria, and eukaryotes) revealed a remarkable statistical paradox, indicating symbiotic origins of eukaryotic cells involving eubacteria. For lipids common to plasma membranes of the three domains, the number of carbon atoms in eubacteria was found to be similar to that in eukaryotes. However, mutually exclusive subsets of same data show exactly the opposite-the number of carbon atoms in lipids of eukaryotes was higher than in eubacteria. This statistical paradox, called Simpson's paradox, was absent for lipids in archaea and for lipids not common to plasma membranes of the three domains. This indicates the presence of interaction(s) and/or association(s) in lipids forming plasma membranes of eubacteria and eukaryotes but not for those in archaea. Further inspection of membrane lipid structures affecting physicochemical properties of plasma membranes provides the first evidence (to our knowledge) on the symbiotic origins of eukaryotic cells based on the "third front" (i.e., lipids) in addition to the growing compositional data from nucleic acids and proteins.

  8. Arsenic and antimony transporters in eukaryotes.

    Science.gov (United States)

    Maciaszczyk-Dziubinska, Ewa; Wawrzycka, Donata; Wysocki, Robert

    2012-01-01

    Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters.

  9. A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

    DEFF Research Database (Denmark)

    Pallisgaard, N; Pedersen, FS; Birkelund, Svend

    1994-01-01

    a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of c......DNA carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

  10. Live (Rose-bengal stained) foraminifera from deep-sea anoxic salt brine in the Eastern Mediterranean: toward understanding limit of life for single-celled eukaryotes (foraminifera)

    Science.gov (United States)

    Kitazato, H.; Ohkawara, N.; Iwasaki, A.; Nomaki, H.; Akoumianaki, I.; Tokuyama, H.

    2012-04-01

    What is a limit of life for the eukaryotes? Eukaryotes are thought to adapt and evolve under oxic environmental conditions. Recently, there are many exceptions for this hypothesis, as many eukaryotes including metazoan groups are found in anoxic environmental conditions. We found many rose-bengal stained foraminifera from a deep-hypersaline anoxic basin (DHAB) in the eastern Mediterranean. During KH06-04 cruise, we conducted oceanographic research at Medée Lake, the largest DHAB, that is located 100km southwest of Crete Island in the eastern Mediterranean. The lake situates at 2920m in water depth. Depth of saline water is 120m in maximum. Both water and sediment samplings were carried out both with Niskin bottles and multiple corer attached to camera watching sampling system at three sites, inside of the lake (CS), the edge of the lake (OMS) and the normal deep-sea floor (RS). Temperature, salinity, and dissolved oxygen concentrations at central saline lake are 15.27 oC, 328PSU, and 0.0 ml/L, respectively. Strong smell of hydrogen sulfide was detected from the lake sediment. Subsamples were conducted for multiple core samples using 3 subcores(φ 2.9cm) from each core tube (φ 8.2cm). Sediment samples were fixed with 4% formalin Rose Bengal solution on board. In laboratory, samples were washed with 32μm sieve. Rose Bengal stained specimens were picked under binocular stereomicroscope (Zeiss Stemi SV11) for surface 0.5cm layer, and identified with inverted microscope (Nikon ECLIPSE TE300). In total, 26 species belonging to 9 genera were identified from three sites. Six species belonging to two genera were identified in the center of the salt brine. Only a few species are common among three sites, even though the numbers of common species were 10 between OMS and RS sites. In DHAB, spherical organic-walled species, such as allogromiid and psammosphaerid, are dominant. In contrast, tube-like chitinous foraminifera, such as Resigella, Conicotheca and Nodellum, are

  11. Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae).

    OpenAIRE

    Cavalier-Smith, T

    2003-01-01

    Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all no...

  12. Control of the cell cycle progression by the MAPK Hog1

    Directory of Open Access Journals (Sweden)

    Josep Clotet

    2013-02-01

    Full Text Available Eukaryotic cells coordinate various intracellular activities in response to environmental stresses, activating an adaptive program to maximize the probability of survival and proliferation. Cells transduce diverse cellular stimuli by multiple mitogen-activated protein kinase (MAPK cascades. MAPK are key signal transduction kinases required to respond to stress. A prototypical member of the MAPK family is the yeast high osmolarity glycerol (Hog1. Activation of Hog1 results in the generation of a set of adaptive responses that leads to the modulation of several aspects of cell physiology that are essential for cell survival, such as gene expression, translation, and morphogenesis. This review focuses on the control of cell cycle progression by Hog1 which is critical for cell survival in response to stress conditions.

  13. The plant cell cycle: Pre-Replication complex formation and controls.

    Science.gov (United States)

    Brasil, Juliana Nogueira; Costa, Carinne N Monteiro; Cabral, Luiz Mors; Ferreira, Paulo C G; Hemerly, Adriana S

    2017-01-01

    The multiplication of cells in all living organisms requires a tight regulation of DNA replication. Several mechanisms take place to ensure that the DNA is replicated faithfully and just once per cell cycle in order to originate through mitoses two new daughter cells that contain exactly the same information from the previous one. A key control mechanism that occurs before cells enter S phase is the formation of a pre-replication complex (pre-RC) that is assembled at replication origins by the sequential association of the origin recognition complex, followed by Cdt1, Cdc6 and finally MCMs, licensing DNA to start replication. The identification of pre-RC members in all animal and plant species shows that this complex is conserved in eukaryotes and, more importantly, the differences between kingdoms might reflect their divergence in strategies on cell cycle regulation, as it must be integrated and adapted to the niche, ecosystem, and the organism peculiarities. Here, we provide an overview of the knowledge generated so far on the formation and the developmental controls of the pre-RC mechanism in plants, analyzing some particular aspects in comparison to other eukaryotes.

  14. Conservation and Variability of Meiosis Across the Eukaryotes.

    Science.gov (United States)

    Loidl, Josef

    2016-11-23

    Comparisons among a variety of eukaryotes have revealed considerable variability in the structures and processes involved in their meiosis. Nevertheless, conventional forms of meiosis occur in all major groups of eukaryotes, including early-branching protists. This finding confirms that meiosis originated in the common ancestor of all eukaryotes and suggests that primordial meiosis may have had many characteristics in common with conventional extant meiosis. However, it is possible that the synaptonemal complex and the delicate crossover control related to its presence were later acquisitions. Later still, modifications to meiotic processes occurred within different groups of eukaryotes. Better knowledge on the spectrum of derived and uncommon forms of meiosis will improve our understanding of many still mysterious aspects of the meiotic process and help to explain the evolutionary basis of functional adaptations to the meiotic program.

  15. 犬SLAM基因真核表达载体的构建及在MDCK细胞中的稳定表达%Construction of Eukaryotic Expressing Vector of SLAM Gene and Establishment of Its Stable Expressing MDCK Cell

    Institute of Scientific and Technical Information of China (English)

    褚秀玲; 苏建青; 江成; 张吉清; 马秀亮

    2012-01-01

    To construction a MDCK cell line stably expressing signalling lymphocyte activation molecules (SLAM). The SLAM gene of cellular receptor of CDV was amplified by RT-PCR from canine peripheral blood lymphocytes. The correctly identified SLAM gene was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/ SLAM. The pcDNA3.1/SLAM was transfected into MDCK cells by Lipofectamine. The stably expressing MDCK cell was screened with DMEM medium under the drug selection of G418. The single clone strain was purified by limiting dilution. The results indicated that the eukaryotic expression vector pcDNA3,l/SLAM was successfully constructed, then the stable expressing MDCK cell line was obtained.%为了构建稳定表达犬信号淋巴细胞激活因子(SLAM)的MDCK细胞系,该研究从犬外周血淋巴细胞中克隆了犬瘟热病毒(Canine distemper virus,CDV)细胞受体SLAM基因,将鉴定正确的SLAM基因插入到高效真核表达载体pcDNA3.1(+)中.采用脂质体转染的方式将重组质粒pcDNA3.1/SLAM转染到MDCK细胞中,采用G418加压筛选及有限稀释法克隆,获取稳定表达SLAM的MDCK细胞株.结果表明,成功构建了SLAM的真核表达载体pcDNA3.1/SLAM,并通过G418筛选获得了稳定表达SLAM的细胞系MDCK.

  16. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  17. RNA Export through the NPC in Eukaryotes.

    Science.gov (United States)

    Okamura, Masumi; Inose, Haruko; Masuda, Seiji

    2015-03-20

    In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

  18. Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

    Science.gov (United States)

    Yusupova, Gulnara; Yusupov, Marat

    2017-03-19

    A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome-the high-eukaryote-specific long ribosomal RNA segments (about 1MDa)-remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved.This article is part of the themed issue 'Perspectives on the ribosome'.

  19. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    Science.gov (United States)

    Blackstone, Neil W.

    2016-01-01

    Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real—the endosymbiosis that led to the mitochondrion is often described as “non-Darwinian” because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious—all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes. PMID:27128953

  20. An Evolutionary Framework for Understanding the Origin of Eukaryotes.

    Science.gov (United States)

    Blackstone, Neil W

    2016-04-27

    Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real-the endosymbiosis that led to the mitochondrion is often described as "non-Darwinian" because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious-all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

  1. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    Directory of Open Access Journals (Sweden)

    Neil W. Blackstone

    2016-04-01

    Full Text Available Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real—the endosymbiosis that led to the mitochondrion is often described as “non-Darwinian” because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious—all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

  2. Molecular mechanisms controlling the cell cycle in embryonic stem cells.

    Science.gov (United States)

    Abdelalim, Essam M

    2013-12-01

    Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.

  3. N1-Guanyl-1,7-Diaminoheptane Sensitizes Estrogen Receptor Negative Breast Cancer Cells to Doxorubicin by Preventing Epithelial-Mesenchymal Transition through Inhibition of Eukaryotic Translation Initiation Factor 5A2 Activation

    Directory of Open Access Journals (Sweden)

    Yu Liu

    2015-08-01

    Full Text Available Background: Approximately 30% of breast cancer does not express the estrogen receptor (ER, which is necessary for endocrine-based therapy approaches. Many studies demonstrated that eukaryotic translation initiation factor 5A2 (eIF5A2 serves as a proliferation-related oncogene in tumorigenic processes. Methods: The present study used cell viability assays, EdU incorporation assays, western blot, and immunofluorescence to explore whether N1-guanyl-1,7-diaminoheptane (GC7, which inhibits eIF5A2 activation, exerts synergistic cytotoxicity with doxorubicin in breast cancer. Results: We found that GC7 enhanced doxorubicin cytotoxicity in ER-negative HCC1937 cells but had little effect in ER-positive MCF-7 and Bcap-37 cells. Administration of GC7 reversed the doxorubicin-induced epithelial-mesenchymal transition (EMT in ER-negative breast cancer cells. Knockdown of eIF5A2 by siRNA inhibited the doxorubicin-induced EMT in ER-negative HCC1937 cells. Conclusion: These data demonstrated that GC7 combination therapy may enhance the therapeutic efficacy of doxorubicin in estrogen negative breast cancer cells by preventing EMT through inhibition of eIF5A2 activation.

  4. Fuel cell with internal flow control

    Science.gov (United States)

    Haltiner, Jr., Karl J.; Venkiteswaran, Arun [Karnataka, IN

    2012-06-12

    A fuel cell stack is provided with a plurality of fuel cell cassettes where each fuel cell cassette has a fuel cell with an anode and cathode. The fuel cell stack includes an anode supply chimney for supplying fuel to the anode of each fuel cell cassette, an anode return chimney for removing anode exhaust from the anode of each fuel cell cassette, a cathode supply chimney for supplying oxidant to the cathode of each fuel cell cassette, and a cathode return chimney for removing cathode exhaust from the cathode of each fuel cell cassette. A first fuel cell cassette includes a flow control member disposed between the anode supply chimney and the anode return chimney or between the cathode supply chimney and the cathode return chimney such that the flow control member provides a flow restriction different from at least one other fuel cell cassettes.

  5. 犬SLAM基因真核表达载体的构建及Vero细胞系转染的研究%Construction of Eukaryotic Expressing Vector of SLAM Gene and its Transient Expression in Vero Cell

    Institute of Scientific and Technical Information of China (English)

    苏建青; 褚秀玲; 张吉清; 马秀亮; 江成

    2012-01-01

    To elucidate the character and functions of cellular receptors of canine distemper virus, the gene SLAM of canine distemper virus' s open reading frame (ORF) was cloned into eukaryotic expression vector pCDNA3.1(+) to generate the recombinant plasmid pcDNA3.1/S.LAM. The pureed plasmid was transected into Vero cells in vitro with Lipofectamine 2000. The transient expression of the SLAM protein was detected by reverse transcription polymerase chain reaction and Westem-blot assay. The results showed that the eukaryotic expression vectors of canine SLAM gene were constructed successfully. The reverse transcription polymerase chain reaction and Western-blot assay confirmed that the protein SLAM was expression in Vero cell. Recombinant SLAM was successfully expressed, which laid foundation for further research on the stable express of SLAM gene in Vero.%为了研究犬瘟热病毒细胞受体SLAM基因的特性和功能.将基因SLAM克隆到真核表达载体pcDNA3.1(+)上,构建真核表达载体pCDNA3.1/SLAM,重组质粒纯化后应用脂质体2000转染到Vero细胞中,通过RT-PCR和免疫印迹方法检测犬SLAM基因在Vero细胞中的转录和表达情况.结果表明:成功构建了表达SLAM基因的真核表达载体,RT-PCR和免疫印迹显示SLAM基因获得表达;pcDNA3.1/SLAM载体的构建为研究犬SLAM基因在Vero细胞中的稳定表达奠定了基础.

  6. Eukaryotic and Prokaryotic Cytoskeletons: Structure and Mechanics

    Science.gov (United States)

    Gopinathan, Ajay

    2013-03-01

    The eukaryotic cytoskeleton is an assembly of filamentous proteins and a host of associated proteins that collectively serve functional needs ranging from spatial organization and transport to the production and transmission of forces. These systems can exhibit a wide variety of non-equilibrium, self-assembled phases depending on context and function. While much recent progress has been made in understanding the self-organization, rheology and nonlinear mechanical properties of such active systems, in this talk, we will concentrate on some emerging aspects of cytoskeletal physics that are promising. One such aspect is the influence of cytoskeletal network topology and its dynamics on both active and passive intracellular transport. Another aspect we will highlight is the interplay between chirality of filaments, their elasticity and their interactions with the membrane that can lead to novel conformational states with functional implications. Finally we will consider homologs of cytoskeletal proteins in bacteria, which are involved in templating cell growth, segregating genetic material and force production, which we will discuss with particular reference to contractile forces during cell division. These prokaryotic structures function in remarkably similar yet fascinatingly different ways from their eukaryotic counterparts and can enrich our understanding of cytoskeletal functioning as a whole.

  7. A new aspect to the origin and evolution of eukaryotes.

    Science.gov (United States)

    Vellai, T; Takács, K; Vida, G

    1998-05-01

    One of the most important omissions in recent evolutionary theory concerns how eukaryotes could emerge and evolve. According to the currently accepted views, the first eukaryotic cell possessed a nucleus, an endomembrane system, and a cytoskeleton but had an inefficient prokaryotic-like metabolism. In contrast, one of the most ancient eukaryotes, the metamonada Giardia lamblia, was found to have formerly possessed mitochondria. In sharp contrast with the traditional views, this paper suggests, based on the energetic aspect of genome organization, that the emergence of eukaryotes was promoted by the establishment of an efficient energy-converting organelle, such as the mitochondrion. Mitochondria were acquired by the endosymbiosis of ancient alpha-purple photosynthetic Gram-negative eubacteria that reorganized the prokaryotic metabolism of the archaebacterial-like ancestral host cells. The presence of an ATP pool in the cytoplasm provided by this cell organelle allowed a major increase in genome size. This evolutionary change, the remarkable increase both in genome size and complexity, explains the origin of the eukaryotic cell itself. The loss of cell wall and the appearance of multicellularity can also be explained by the acquisition of mitochondria. All bacteria use chemiosmotic mechanisms to harness energy; therefore the periplasm bounded by the cell wall is an essential part of prokaryotic cells. Following the establishment of mitochondria, the original plasma membrane-bound metabolism of prokaryotes, as well as the funcion of the periplasm providing a compartment for the formation of different ion gradients, has been transferred into the inner mitochondrial membrane and intermembrane space. After the loss of the essential function of periplasm, the bacterial cell wall could also be lost, which enabled the naked cells to establish direct connections among themselves. The relatively late emergence of mitochondria may be the reason why multicellularity evolved so

  8. How eukaryotic genes are transcribed

    OpenAIRE

    Venters, Bryan J; Pugh, B. Franklin

    2009-01-01

    Regulation of eukaryotic gene expression is far more complex than one might have imagined thirty years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: 1) a well-defined organization of nucleosomes and modification states at most genes, 2) regulatory networks of sequence-specific ...

  9. Prokaryotes Versus Eukaryotes: Who is Hosting Whom?

    Science.gov (United States)

    Tellez, Guillermo

    2014-01-01

    Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals' actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a "forgotten organ," functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short-chain fatty acids), a process, which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system. Despite these important effects, the mechanisms by which the gut microbial community influences the host's biology remain almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes, which encourage us to postulate: who is hosting whom?

  10. Prokaryotes versus Eukaryotes: Who is hosting whom?

    Directory of Open Access Journals (Sweden)

    Guillermo eTellez

    2014-10-01

    Full Text Available Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals’ actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a ‘forgotten organ’, functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short chain fatty acids, a process which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system,. Despite these important effects, the mechanisms by which the gut microbial community influences the host’s biology remains almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes which encourage us to postulate: Who is

  11. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    Science.gov (United States)

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  12. Cell Biology of Prokaryotic Organelles

    OpenAIRE

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-01-01

    Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-...

  13. Asymmetric cell division during T cell development controls downstream fate

    Science.gov (United States)

    Pham, Kim; Shimoni, Raz; Charnley, Mirren; Ludford-Menting, Mandy J.; Hawkins, Edwin D.; Ramsbottom, Kelly; Oliaro, Jane; Izon, David; Ting, Stephen B.; Reynolds, Joseph; Lythe, Grant; Molina-Paris, Carmen; Melichar, Heather; Robey, Ellen; Humbert, Patrick O.; Gu, Min

    2015-01-01

    During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal. PMID:26370500

  14. Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

    Directory of Open Access Journals (Sweden)

    Andreeva Alexandra V

    2004-11-01

    Full Text Available Abstract Background In eukaryotes, PPP (protein phosphatase P family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases. Results We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a

  15. Arsenic transport in prokaryotes and eukaryotic microbes.

    Science.gov (United States)

    Rosen, Barry P; Tamás, Markus J

    2010-01-01

    Aquaporins (AQPs) and aquaglyceroporins facilitate transport of a broad spectrum of substrates such as water, glycerol and other small uncharged solutes. More recently, AQPs ave also been shown to facilitate diffusion of metalloids such as arsenic (As) and antimony (Sb). At neutral pH, the trivalent forms of these metalloids are structurally similar to glycerol and hence they can enter cells through AQPs. As- and Sb-containing compounds are toxic to cells, yet both metalloids are used as chemotherapeutic agents for treating acute promyelocytic leukemia and diseases caused by protozoan parasites. In this chapter, we will review the role of AQPs and other proteins in metalloid transport in prokaryotes and eukaryotic microbes.

  16. Developmental control of cell division

    NARCIS (Netherlands)

    Boxem, M. (Mike)

    2002-01-01

    During development of multicellular organisms, cell divisions need to be coordinated with the developmental program of the entire organism. Although the mechanisms that drive cells through the division cycle are well understood, very little is known about the pathways that link extracellular signals

  17. Developmental control of cell division

    NARCIS (Netherlands)

    Boxem, M. (Mike)

    2002-01-01

    During development of multicellular organisms, cell divisions need to be coordinated with the developmental program of the entire organism. Although the mechanisms that drive cells through the division cycle are well understood, very little is known about the pathways that link extracellular signals

  18. High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.

    Science.gov (United States)

    Torresi, J; Meanger, J; Lambert, P; Li, F; Locarnini, S A; Anderson, D A

    1997-12-01

    The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

  19. Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation

    OpenAIRE

    Simon,Anne; Maletz, Sibylle X; Hollert, Henner; Schäffer, Andreas; Maes, Hanna M

    2014-01-01

    To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L...

  20. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  1. Process for control of cell division

    Science.gov (United States)

    Cone, C. D., Jr. (Inventor)

    1977-01-01

    A method of controlling mitosis of biological cells was developed, which involved inducing a change in the intracellular ionic hierarchy accompanying the cellular electrical transmembrane potential difference (Esubm) of the cells. The ionic hierarchy may be varied by imposing changes on the relative concentrations of Na(+), K(+) and Cl(-), or by directly imposing changes in the physical Esubm level across the cell surface.

  2. Cdc42-mediated tubulogenesis controls cell specification

    DEFF Research Database (Denmark)

    Kesavan, Gokul; Sand, Fredrik Wolfhagen; Greiner, Thomas Uwe

    2009-01-01

    Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled......, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later...... for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors. For a video summary of this article, see the PaperFlick file with the Supplemental Data...

  3. A new inhibitor of apoptosis from vaccinia virus and eukaryotes.

    NARCIS (Netherlands)

    Gubser, C.; Bergamaschi, D.; Hollinshead, M.; Lu, X.; Kuppeveld, F.J.M. van; Smith, G.L.

    2007-01-01

    A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAA

  4. Regulation of eukaryotic DNA replication and nuclear structure

    Institute of Scientific and Technical Information of China (English)

    WUJIARUI

    1999-01-01

    In eukaryote,nuclear structure is a key component for the functions of eukaryotic cells.More and more evidences show that the nuclear structure plays important role in regulating DNA replication.The nuclear structure provides a physical barrier for the replication licensing,participates in the decision where DNA replication initiates,and organizes replication proteins as replication factory for DNA replication.Through these works,new concepts on the regulation of DNA replication have emerged,which will be discussed in this minireview.

  5. Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation.

    Science.gov (United States)

    Simon, Anne; Maletz, Sibylle X; Hollert, Henner; Schäffer, Andreas; Maes, Hanna M

    2014-01-01

    To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L, and 3.13 to 50 mg CNT/L + 1% TCC (percentage relative to carbon nanotubes concentration), respectively. Triclocarban is a high-production volume chemical that is widely used as an antimicrobial compound and is known for its toxicity, hydrophobicity, endocrine disruption, bioaccumulation potential, and environmental persistence. Carbon nanotubes are known to interact with hydrophobic organic compounds. Therefore, triclocarban was selected as a model substance to examine mixture toxicity in this study. The influence of multiwalled carbon nanotubes and triclocarban on various toxicological endpoints was specified: neither cytotoxicity nor endocrine disruption could be observed after exposure of the three cell lines to carbon nanotubes, but the nanomaterial caused intracellular generation of reactive oxygen species in all cell types. For TCC on the other hand, cell vitality of 80% could be observed at a concentration of 2.1 mg/L for treated RTL-W1 cells. A decrease of luciferase activity in the ER Calux assay at a triclocarban concentration of 125 μg/L and higher was observed. This effect was less pronounced when multiwalled carbon nanotubes were present in the medium. Taken together, these results demonstrate that multiwalled carbon nanotubes induce the production of reactive oxygen species in RTL-W1, T47Dluc, and H295R cells, reveal no cytotoxicity, and reduce the bioavailability and toxicity of the biocide triclocarban.

  6. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  7. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

    Science.gov (United States)

    Denks, Kärt; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

  8. Expression ofpprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells%耐辐射奇球菌pprI和pprA基因在真核细胞293T中的表达

    Institute of Scientific and Technical Information of China (English)

    肖潇; 马云; 肖方竹; 唐艳; 杨奇; 黄波; 唐旻; 何淑雅

    2016-01-01

    To construct fluorescence expression plasmids pEGFP-N1-pprA and pDsRed1-N1-flag-pprI, pGADT-7-pprA and pet28a-pprI plasmid constructed at an earlier laboratory stage was used as the template, and Lipofectamine 2000 was employed to transfect two recombinant vectors into 293T cells. A fluorescent microscope was used for Fluorescence observation, and Western blot was employed to examine the expression of the fusion protein. Double digestions and the agarose gel electrophoresis showed that target bands appeared at 4700 bp and 1000 bp, 4800 bp and 1100 bp. No apparent frameshift mutation occurred as shown in the sequencing results. The fluorescent microscopy showed red and green phosphors; the Western blot results indicated protein expression at 65 kDa and 60 kDa. pDsRed1-N1-flag-pprI and pEGFP-N1-pprA were successfully constructed to express proteins for eukaryotic cells 293T in vitro. The results indicated that prokaryotic genespprA and pprI could co-express proteins in eukaryotic cells successfully, and laid a foundation for the interaction and synergism ofpprA,pprI, and their products in the regulation network of radiation verified by DR, enhancing the radiation resistance of eukaryotic cells.%以pGADT-7-pprA、pet28a-pprI载体为模板,构建pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体,脂质体2000介导将两个重组载体共转入293T细胞.双酶切及琼脂糖凝胶电泳显示在4700、1000 bp处与4800、1100 bp处出现目的条带.测序结果显示构建序列与模板序列一致,氨基酸序列100%正确.荧光显微镜下见到红色和绿色荧光;Western blot结果显示在不同检测水平65 kDa及60 kDa大小处有融合蛋白表达.结果提示pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体构建成功,并在离体293T细胞中共同表达蛋白.证明原核基因pprI、pprA能够在真核细胞中共表达,为后续实验验证DR菌高抗性基因pprA、pprI及其产物在辐射调控网络中的相互作用和协同作用、

  9. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-I2 and IFN-γ,and may be play an important part in the process of controlling acute phase of toxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH Ⅰ and EcoR Ⅰ restriction enzyme site. The cDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector.After cloned, the vector was digested with BamH Ⅰ and EcoR Ⅰ and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAXI-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasma gondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用.本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHⅠ和EcoRⅠ酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因.PCR产物连接到pMD18-T克隆载体.用限

  10. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-12 and IFN-γ and may be play an important part in the process of controlling acute phase oftoxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH I and EcoR I restriction enzyme site. The eDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector. After cloned, the vector was digested with BamH I and EcoR I and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAX1-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasmngondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载

  11. Engineering controlled mammalian type O-Glycosylation in plant cells

    DEFF Research Database (Denmark)

    Yang, Zhang; Drew, Damian Paul; Jørgensen, Bodil

    2011-01-01

    Human mucins are large heavily O-glycosylated glycoproteins (>200 kDa), which account for the majority of proteins in mucus layers that e.g. hydrate, lubricate and protect cells from proteases as well as from pathogens. O-linked mucin glycans are truncated in many cancers, yielding truncated cancer...... specific glyco-peptide epitopes, such as the Tn epitope (GalNAc sugar attached to either Serine or Threonine), which are antigenic to the immune system. In the present study, we have identified plant cells as the only eukaryotic cells without mammalian type O-glycosylation or competing (for sites) O...

  12. Eukaryotic protein production in designed storage organelles

    Directory of Open Access Journals (Sweden)

    Saloheimo Markku

    2009-01-01

    Full Text Available Abstract Background Protein bodies (PBs are natural endoplasmic reticulum (ER or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells. Results Various Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain. Conclusion The Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

  13. 沙眼衣原体ompA基因克隆及其在真核细胞中的表达%Cloning of ompA gene from Chlamydia trachomatis and its expression in eukaryotic cell

    Institute of Scientific and Technical Information of China (English)

    李忠玉; 吴移谋; 陈超群; 余敏君

    2004-01-01

    目的克隆D型沙眼衣原体(Ct)ompA基因,构建真核表达重组质粒,转染真核细胞,为核酸疫苗的研制作准备.方法用PCR技术从D型Ct基因组DNA中扩增ompA基因片段,重组入pUCm-T克隆载体.将pUCm-T/ompA中的ompA外源基因片段经酶切、连接等反应,亚克隆入pcDNA3.1真核表达载体,进行序列分析和酶切鉴定后,运用脂质体将重组体pcDNA3.1/ompA转染HeLa细胞,免疫组化法观察目的的基因的表达.结果从D型Ct基因组DNA中扩增出特异的ompA基因片段;序列测定证实与GenBank登陆的D型Ct一致;重组质粒pcDNA3.1/ompA在HeLa中获得表达.结论Ct ompA基因能够在体外真核细胞表达,为进一步研究Ct致病机制及DNA疫苗的研究提供理论依据.%Objective: To clone and construct the recombinant plasmid containing ompA gene from Chlamydia trachomatis, and transfect it into mammalian cells to express the major outer membrane protein (MOMP). Methods: Amplifed from the genomic DNA of Chlamydia trachomatis using polymerase chain reaction (PCR), ompA gene was inserted into cloning vector pUCm-T. The inserted ompA gene was subcloned to pcDNA3.1 eukaryotic expression vector by linking reactions. After identifying by sequencing and restrictive enzymes digestion. The recombinant plasmid was transfected into HeLa cells using Liposome. Results: The specific gene fragment about 1.2 kb was successfully amplified. The DNA sequence of ompA gene was found to be the same as the nucleotide sequence published by GenBank. Immunocytochemistry analysis showed that om pA gene was expressed in HeLa cells and located in cytoplasm. Conclusion: OmpA gene can be expressed in eukaryotic system which lay the foundation for studing the pathogenic mechanism and the development of the Chlamydia trachomatis vaccine against this pathogen.

  14. Sjcb2 DNA疫苗的构建及其在真核细胞中的表达%Construction of Sjcb2 DNA vaccine and its expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    胡永轩; 肖建华; 黄家芳; 杨秋林

    2006-01-01

    Objective To clone and construct the recombinant plasmid containing cathepsin B endopeptidase of Schistosomajaponicum (Sjcb2) and transfer it into mammalian cells to express Cathepsin B endopeptidase protein. Methods By polymerasechain reaction(PCR) tenique, Sjcb2 was amplified from the constructed recombinant plasmid pBC SK +/Sjcb2. Followly Sjcb2 wasinserted into cloning vector pUCm - T. Then Sjcb2 was subcloned to the eukaryotic expression vector pcDNA3.1 ( + ) by linking reactions. After identifying it by PCR,restrietive enzymes digestion and DNA sequencing, the recombinant plamid was transfected into HeLa cells using electroporation, and analyze the expression of the recombinant protein by indirect immunofluorescence assay. Results The specific gene fragment about 1047bp was successfully amplified. The DNA vaccine of Sjcb2 was successfully constructed.Indirect immunofluorescence assay showed that Sjcb2 was expressed in HeLa cells and located in cytoplasm. Conclusion Sjcb2gene can be expressed in eukaryotic system, which lay the foundation for studying the pathogenic mechanism and the development ofthe Sjcb2 DNA vaccine against Schitosomiasis.%目的克隆日本血吸虫组织蛋白酶B基因,构建真核表达重组质粒,转染真核细胞,观察其表达情况.方法用PCR技术从已构建好的重组质粒pBC SK+/Sjcb2中扩增出Sjcb2基因片段,重组入克隆载体pUCm-T.再将Sjcb2基因片段亚克隆入真核表达载体pcDNA3.1(+).进行PCR、双酶切和DNA序列鉴定后,运用电穿孔技术将重组体pcDNA3.1(+)/Sjcb2转染HeLa细胞,间接免疫荧光技术观察目的基因的表达. 结果成功扩增出Sjcb2基因片段并构建DNA疫苗;重组质粒在HeLa细胞中获得表达.结论Sjcb2基因能够在体外真核细胞中表达.

  15. Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.

    Science.gov (United States)

    Materna, Stefan C; Marwan, Wolfgang

    2005-02-01

    We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

  16. Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle

    NARCIS (Netherlands)

    Papagiannakis, Alexandros; Niebel, Bastian; Wit, Ernst C.; Heinemann, Matthias

    2017-01-01

    Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic trigg

  17. Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle

    NARCIS (Netherlands)

    Papagiannakis, Alexandros; Niebel, Bastian; Wit, Ernst C; Heinemann, Matthias

    2017-01-01

    Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic

  18. Eukaryotic and prokaryotic contributions to colonic hydrogen sulfide synthesis.

    Science.gov (United States)

    Flannigan, Kyle L; McCoy, Kathy D; Wallace, John L

    2011-07-01

    Hydrogen sulfide (H(2)S) is an important modulator of many aspects of digestive function, both in health and disease. Colonic tissue H(2)S synthesis increases markedly during injury and inflammation and appears to contribute to resolution. Some of the bacteria residing in the colon can also produce H(2)S. The extent to which bacterial H(2)S synthesis contributes to what is measured as colonic H(2)S synthesis is not clear. Using conventional and germ-free mice, we have delineated the eukaryotic vs. prokaryotic contributions to colonic H(2)S synthesis, both in healthy and colitic mice. Colonic tissue H(2)S production is entirely dependent on the presence of the cofactor pyridoxal 5'-phosphate (vitamin B(6)), while bacterial H(2)S synthesis appears to occur independent of this cofactor. As expected, approximately one-half of the H(2)S produced by feces is derived from eukaryotic cells. While colonic H(2)S synthesis is markedly increased when the tissue is inflamed, and, in proportion to the extent of inflammation, fecal H(2)S synthesis does not change and tissue granulocytes do not appear to be the source of the elevated H(2)S production. Rats fed a B vitamin-deficient diet for 6 wk exhibited significantly diminished colonic H(2)S synthesis, but fecal H(2)S synthesis was not different from that of rats on the control diet. Our results demonstrate that H(2)S production by colonic bacteria does not contribute significantly to what is measured as colonic tissue H(2)S production, using the acetate trapping assay system employed in this study.

  19. A general strategy to construct small molecule biosensors in eukaryotes

    Science.gov (United States)

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-01-01

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.10606.001 PMID:26714111

  20. Uniting sex and eukaryote origins in an emerging oxygenic world

    Directory of Open Access Journals (Sweden)

    Gross Jeferson

    2010-08-01

    Full Text Available Abstract Background Theories about eukaryote origins (eukaryogenesis need to provide unified explanations for the emergence of diverse complex features that define this lineage. Models that propose a prokaryote-to-eukaryote transition are gridlocked between the opposing "phagocytosis first" and "mitochondria as seed" paradigms, neither of which fully explain the origins of eukaryote cell complexity. Sex (outcrossing with meiosis is an example of an elaborate trait not yet satisfactorily addressed in theories about eukaryogenesis. The ancestral nature of meiosis and its dependence on eukaryote cell biology suggest that the emergence of sex and eukaryogenesis were simultaneous and synergic and may be explained by a common selective pressure. Presentation of the hypothesis We propose that a local rise in oxygen levels, due to cyanobacterial photosynthesis in ancient Archean microenvironments, was highly toxic to the surrounding biota. This selective pressure drove the transformation of an archaeal (archaebacterial lineage into the first eukaryotes. Key is that oxygen might have acted in synergy with environmental stresses such as ultraviolet (UV radiation and/or desiccation that resulted in the accumulation of reactive oxygen species (ROS. The emergence of eukaryote features such as the endomembrane system and acquisition of the mitochondrion are posited as strategies to cope with a metabolic crisis in the cell plasma membrane and the accumulation of ROS, respectively. Selective pressure for efficient repair of ROS/UV-damaged DNA drove the evolution of sex, which required cell-cell fusions, cytoskeleton-mediated chromosome movement, and emergence of the nuclear envelope. Our model implies that evolution of sex and eukaryogenesis were inseparable processes. Testing the hypothesis Several types of data can be used to test our hypothesis. These include paleontological predictions, simulation of ancient oxygenic microenvironments, and cell biological

  1. Uniting sex and eukaryote origins in an emerging oxygenic world.

    Science.gov (United States)

    Gross, Jeferson; Bhattacharya, Debashish

    2010-08-23

    Theories about eukaryote origins (eukaryogenesis) need to provide unified explanations for the emergence of diverse complex features that define this lineage. Models that propose a prokaryote-to-eukaryote transition are gridlocked between the opposing "phagocytosis first" and "mitochondria as seed" paradigms, neither of which fully explain the origins of eukaryote cell complexity. Sex (outcrossing with meiosis) is an example of an elaborate trait not yet satisfactorily addressed in theories about eukaryogenesis. The ancestral nature of meiosis and its dependence on eukaryote cell biology suggest that the emergence of sex and eukaryogenesis were simultaneous and synergic and may be explained by a common selective pressure. We propose that a local rise in oxygen levels, due to cyanobacterial photosynthesis in ancient Archean microenvironments, was highly toxic to the surrounding biota. This selective pressure drove the transformation of an archaeal (archaebacterial) lineage into the first eukaryotes. Key is that oxygen might have acted in synergy with environmental stresses such as ultraviolet (UV) radiation and/or desiccation that resulted in the accumulation of reactive oxygen species (ROS). The emergence of eukaryote features such as the endomembrane system and acquisition of the mitochondrion are posited as strategies to cope with a metabolic crisis in the cell plasma membrane and the accumulation of ROS, respectively. Selective pressure for efficient repair of ROS/UV-damaged DNA drove the evolution of sex, which required cell-cell fusions, cytoskeleton-mediated chromosome movement, and emergence of the nuclear envelope. Our model implies that evolution of sex and eukaryogenesis were inseparable processes. Several types of data can be used to test our hypothesis. These include paleontological predictions, simulation of ancient oxygenic microenvironments, and cell biological experiments with Archaea exposed to ROS and UV stresses. Studies of archaeal conjugation

  2. Alternative splicing: a pivotal step between eukaryotic transcription and translation.

    Science.gov (United States)

    Kornblihtt, Alberto R; Schor, Ignacio E; Alló, Mariano; Dujardin, Gwendal; Petrillo, Ezequiel; Muñoz, Manuel J

    2013-03-01

    Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

  3. Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells.

    Science.gov (United States)

    Álvarez-Fraga, Laura; Pérez, Astrid; Rumbo-Feal, Soraya; Merino, María; Vallejo, Juan Andrés; Ohneck, Emily J; Edelmann, Richard E; Beceiro, Alejandro; Vázquez-Ucha, Juan C; Valle, Jaione; Actis, Luis A; Bou, Germán; Poza, Margarita

    2016-05-18

    Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii.

  4. Controllability analysis of decentralised linear controllers for polymeric fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Serra, Maria; Aguado, Joaquin; Ansede, Xavier; Riera, Jordi [Institut de Robotica i Informatica Industrial, Universitat Politecnica de Catalunya - Consejo Superior de Investigaciones Cientificas, C. Llorens i Artigas 4, 08028 Barcelona (Spain)

    2005-10-10

    This work deals with the control of polymeric fuel cells. It includes a linear analysis of the system at different operating points, the comparison and selection of different control structures, and the validation of the controlled system by simulation. The work is based on a complex non linear model which has been linearised at several operating points. The linear analysis tools used are the Morari resiliency index, the condition number, and the relative gain array. These techniques are employed to compare the controllability of the system with different control structures and at different operating conditions. According to the results, the most promising control structures are selected and their performance with PI based diagonal controllers is evaluated through simulations with the complete non linear model. The range of operability of the examined control structures is compared. Conclusions indicate good performance of several diagonal linear controllers. However, very few have a wide operability range. (author)

  5. Abundance of eukaryotic microbes in the deep subtropical North Atlantic

    NARCIS (Netherlands)

    Morgan-Smith, D.; Herndl, G.J.; van Aken, H.M.; Bochdansky, A.B.

    2011-01-01

    The meso- and bathypelagic ocean comprises the largest habitat on earth, yet we know very little about the distribution and activity of protists in this environment. These small eukaryotes are responsible for controlling bacterial abundance in the surface ocean and are major players in the material

  6. Cloning and Expression of Spider Dragline Silk Protein Gene in Escherichia coli and Eukaryotic Cells%蜘蛛拖牵丝蛋白基因的克隆与表达

    Institute of Scientific and Technical Information of China (English)

    刘丹梅; 王芬; 李文利

    2011-01-01

    The extreme tensile strength and toughness of spider silk make it a superior candidate for a wide range of medical and industrial applications. In this study, a 837 bp fragment which encode dragline silk gene (ASP) was cloned from the genome of spider (Araneus ventricosus) and subcloned into pGEX-6p-l prokaryotic expression vector and pGFP-N2 eukaryotic expression vector, named pASG and pASN respectively. By induction at 16 ℃ for 24 h, the fusion protein GST-ASP was successfully expressed and characterized by Westem blotting with anti-GST antibodies. The fusion proteins ASP-GFP was also successfully expressed in insect sf9 cells by the detection of bright green fluorescence. That indicated the ASP gene was corrected expressed in E. coli and eukaryotic cells, respectively. This study provides a beneficial attempt to exploiting the pathway of producting spider silk protein by genetic engineering.%蜘蛛丝具有极高的强度和韧度,工业和医学应用价值很高,但由于蜘蛛的不可驯养性使其应用受到限制.因此,本文尝试利用基因工程的方法获得蛛丝蛋白的表达.我们利用巢式PCR技术从大腹圆蛛Araneus ventricosus基因组中克隆了长度为837 bp的拖牵丝蛋白基因(ASP),并分别将其构建至原核表达载体pGEX-6p-1和真核表达载体pGFP-N2上,分别命名为pASG和pASN.pASG在大肠杆菌中16℃下24 h诱导表达后,经蛋白质印迹证明成功地表达了GST-ASP融合蛋白;pASN转染昆虫sf9细胞48 h后观察到了绿色荧光蛋白GFP的表达,表明ASP基因在大肠杆菌和真核细胞中分别得到了正确表达.本研究为利用基因工程的方法开发蛛丝蛋白的生产途径提供了有益的尝试.

  7. Photosensitive chitosan to control cell attachment.

    Science.gov (United States)

    Cheng, Nan; Cao, Xudong

    2011-09-01

    An approach to control cell adhesion using a photocleavable molecule on chitosan has been developed and studied. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) was introduced into chitosan to control the surface properties. The two UV illuminations with a photomask controlled the cleavage of NVOC and the presentation of deprotected amines on one chitosan surface spatially and temporally. The following immobilizations of cell repulsive poly(ethylene glycol) after the first illumination and cell adhesive sequence Arg-Gly-Asp-Ser (RGDS) after the second illumination on the surface helped create surface heterogeneity. Fourier transform infrared spectroscopy (FTIR), water contact angle, and UV-visible spectroscopy were used to characterize the surfaces and photoactivation during the process. To study the cell attachment and morphology on our designed surfaces, NIH/3T3 fibroblast cell was used. Cell number and morphology on the surfaces were investigated. The cell study demonstrated the feasibility of the surfaces on the control of cell adhesion and the formation of cell patterns by UV illuminations and the following immobilizations of different biomolecules. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  8. Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

    Directory of Open Access Journals (Sweden)

    Nguyen-Hieu Tung

    2012-09-01

    Full Text Available Abstract Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p  Conclusion In this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

  9. What Entamoeba histolytica and Giardia lamblia tell us about the evolution of eukaryotic diversity

    Indian Academy of Sciences (India)

    J Samuelson

    2002-11-01

    Entamoeba histolytica and Giardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions, early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.

  10. Genetic and epigenetic factors affecting meiosis induction in eukaryotes revealed in paramecium research.

    Science.gov (United States)

    Prajer, Małgorzata

    2008-01-01

    This review presents studies of the induction of meiosis undertaken on the ciliate Paramecium, a unicellular model eukaryotic organism. Meiosis in Paramecium, preceding the process of fertilization, appears in starved cells after passing a defined number of divisions (cell generations), starting from the last fertilization. Investigations were performed on clones of cells entering autogamy, a self-fertilization process. Genetic as well as epigenetic factors, i.e. endo- and exogenous factors, affecting the induction ofmeiosis and changing the duration of the interautogamous interval (IAI), were analyzed. The results show that: (1) Meiosis induction is controlled genetically by the somatic macronucleus. However, besides the nuclear factors, the cytoplasmic protein immaturin also affects this process (Haga & Hiwatashi 1981); (2) Epigenetic factors, such as non-genetically disturbed cytoskeleton structures and changes in the cell architecture observed in doublet Paramecium cells, exert internal mechanical stress (Ingber 2003), which constitutes the endogenous impulse accelerating meiosis; (3) Mild osmotic stress, acting as an exogenous factor, can initiate the specific MAP kinases signaling pathway resulting in earlier meiosis induction, as in other unicellular eukaryotes (Seet & Pawson 2004).

  11. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

    Science.gov (United States)

    Zhang, Mengmeng; Gill, Gordon N; Zhang, Yan

    2010-01-01

    In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  12. A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

    Science.gov (United States)

    Ilk, Nicola; Küpcü, Seta; Moncayo, Gerald; Klimt, Sigrid; Ecker, Rupert C; Hofer-Warbinek, Renate; Egelseer, Eva M; Sleytr, Uwe B; Sára, Margit

    2004-04-15

    The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

  13. 大鼠FasL全长cDNA真核质粒的构建及转染HepG2细胞体外诱导细胞凋亡的作用%Construction of eukaryotic plasmids of rat FasL full length cDNA and role of FasL transfection in induction of in vitro apoptosis of HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    许彪; 胡瑾华; 徐东平; 李晓东; 刘妍; 陈婧; 王业东; 王慧芬

    2011-01-01

    Objective To construct the eukaryotic expression vector and observe the effect of FasL gene transfection on induction of apoptosis of HepG2 cells. Methods RT-PCR and TA cloning technique were used to amplify the rFasL full length cDNA from rat testis cells, and then a eukaryotic expression vector pDC315 containing rFasL cDNA was constructed. Plasmid was transfected into HepG2 cells. The cells were harvested after 48h to identify the expression of plasmids by RT-PCR and Western blot. Meanwhile, two groups of HepG2 cells were respectively transfected with pDC315 plasmid as a plasmid control group and with H2O as a blank control group. After 48h of culture, the three groups of cells were counted, compared and analyzed. Results The sequence of cloned rFasL cDNA was consistent with that in GenBank. The HepG2 cells transfected with rFasL eukaryotic expression vector could express rFasL mRNA and protein. A large number of HepG2 cells died in the pDC315-rFasL experimental group. Conclusion The rFasL cDNA can be cloned and its eukaryotic expression vector can be constructed, indicating that rFasL gene can be expressed in HepG2 cells transfected with pDC315-rFasL. HepG2 cells expressing FasL induce apoptosis of neighbor-or auto-HepG2 cells, leading to their death.%目的 观察HepG2细胞过表达FasL介导细胞凋亡的作用.方法 从大鼠睾丸细胞中扩增出rFasL全长eDNA,亚克隆到T载体中,再克隆到pDC315载体中.转染HepG2细胞后用RT-PCR、Wester blot检测rFasL mRNA和蛋白表达,培养48h后收集细胞计数,进行比较分析.结果 rFasL cDNA序列与其在Genbank中的序列完全一致.rFasL真核表达载体转染HepG2细胞后能表达rFasL mRNA和蛋白;转染pDC315一rFasL的实验组HepG2细胞大最死亡.结论 成功克隆了rFasL基因并构建其真核表达载体,证明能有效表达于HepG2中,表达FasL的HepG2细胞通过Fas-FasL结合介导周围及自身表达Fas的HepG2细胞凋亡.

  14. MadR1, a Mycobacterium tuberculosis cell cycle stress response protein that is a member of a widely conserved protein class of prokaryotic, eukaryotic and archeal origin.

    Science.gov (United States)

    Crew, Rebecca; Ramirez, Melissa V; England, Kathleen; Slayden, Richard A

    2015-05-01

    Stress-induced molecular programs designed to stall division progression are nearly ubiquitous in bacteria, with one well-known example being the participation of the SulA septum inhibiting protein in the SOS DNA damage repair response. Mycobacteria similarly demonstrate stress-altered growth kinetics, however no such regulators have been found in these organisms. We therefore set out to identify SulA-like regulatory proteins in Mycobacterium tuberculosis. A bioinformatics modeling-based approach led to the identification of rv2216 as encoding for a protein with weak similarity to SulA, further analysis distinguished this protein as belonging to a group of uncharacterized growth promoting proteins. We have named the mycobacterial protein encoded by rv2216 morphology altering division regulator protein 1, MadR1. Overexpression of madR1 modulated cell length while maintaining growth kinetics similar to wild-type, and increased the proportion of bent or V-form cells in the population. The presence of MadR1-GFP at regions of cellular elongation (poles) and morphological differentiation (V-form) suggests MadR1 involvement in phenotypic heterogeneity and longitudinal cellular growth. Global transcriptional analysis indicated that MadR1 functionality is linked to lipid editing programs required for growth and persistence. This is the first report to differentiate the larger class of these conserved proteins from SulA proteins and characterizes MadR1 effects on the mycobacterial cell.

  15. Controlled surface chemistries and quantitative cell response

    Science.gov (United States)

    Plant, Anne L.

    2002-03-01

    Living cells experience a large number of signaling cues from their extracellular matrix. As a result of these inputs, a variety of intracellular signaling pathways are apparently initiated simultaneously. The vast array of alternative responses that result from the integration of these inputs suggests that it may be reasonable to look for cellular response not as an 'on' or 'off' condition but as a distribution of responses. A difficult challenge is to determine whether variations in responses from individual cells arise from the complexity of intracellular signals or are due to variations in the cell culture environment. By controlling surface chemistry so that every cell 'sees' the same chemical and physical environment, we can begin to assess how the distribution of cell response is affected strictly by changes in the chemistry of the cell culture surface. Using the gene for green fluorescent protein linked to the gene for the promoter of the extracellular matrix protein, tenascin, we can easily probe the end product in a signaling pathway that is purported to be linked to surface protein chemistry and to cell shape. Cell response to well-controlled, well-characterized, and highly reproducible surfaces prepared using soft lithography techniques are compared with more conventional ways of preparing extracellular matrix proteins for cell culture. Using fluorescence microscopy and image analysis of populations of cells on these surfaces, we probe quantitatively the relationship between surface chemistry, cell shape and variations in gene expression endpoint.

  16. Infrared-Controlled Welding of Solar Cells

    Science.gov (United States)

    Paulson, R.; Finnell, S. E.; Decker, H. J.; Hodor, J. R.

    1982-01-01

    Proposed apparatus for welding large arrays of solar cells to flexible circuit substrates would sense infrared emission from welding spot. Emission would provide feedback for control of welding heat. Welding platform containing optical fibers moves upward through slots in movable holding fixture to contact solar cells. Fibers pick up infrared radiation from weld area.

  17. Cloning and subcellular location of an arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, S.U.; Bar-Peled, M.; Raikhel, N.V. [Michigan State Univ., East Lansing, MI (United States)

    1997-05-01

    Many receptors involved in clathrin-mediated protein transport through the endocytic and secretary pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. in addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell. 87 refs., 7 figs.

  18. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

    Science.gov (United States)

    Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

  19. Exposure of E. coli to DNA-Methylating Agents Impairs Biofilm Formation and Invasion of Eukaryotic Cells via Down Regulation of the N-Acetylneuraminate Lyase NanA

    Science.gov (United States)

    Di Pasquale, Pamela; Caterino, Marianna; Di Somma, Angela; Squillace, Marta; Rossi, Elio; Landini, Paolo; Iebba, Valerio; Schippa, Serena; Papa, Rosanna; Selan, Laura; Artini, Marco; Palamara, Anna Teresa; Duilio, Angela

    2016-01-01

    DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS) by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA) involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC) strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions. PMID:26904018

  20. Exposure of E. coli to DNA-Methylating Agents Impairs Biofilm Formation and Invasion of Eukaryotic Cells via Down Regulation of the N-Acetylneuraminate Lyase NanA.

    Science.gov (United States)

    Di Pasquale, Pamela; Caterino, Marianna; Di Somma, Angela; Squillace, Marta; Rossi, Elio; Landini, Paolo; Iebba, Valerio; Schippa, Serena; Papa, Rosanna; Selan, Laura; Artini, Marco; Palamara, Anna Teresa; Palamara, Annateresa; Duilio, Angela

    2016-01-01

    DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS) by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA) involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC) strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

  1. Exposure of E. coli to DNA-methylating agents impairs biofilm formation and invasion of eukaryotic cells via down regulation of the N-acetylneuraminate lyase NanA

    Directory of Open Access Journals (Sweden)

    Pamela eDi Pasquale

    2016-02-01

    Full Text Available DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analogue acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

  2. Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

    Science.gov (United States)

    Knight, John R P; Bastide, Amandine; Roobol, Anne; Roobol, Jo; Jackson, Thomas J; Utami, Wahyu; Barrett, David A; Smales, C Mark; Willis, Anne E

    2015-01-15

    Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

  3. Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes.

    Science.gov (United States)

    Daza, Rafael; Cruces, Julia; Arroyo-Hernández, María; Marí-Buyé, Núria; De la Fuente, Mónica; Plaza, Gustavo R; Elices, Manuel; Pérez-Rigueiro, José; Guinea, Gustavo V

    2015-03-19

    The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.

  4. [Role of eukaryotic translation initiation factor 4G in tumor].

    Science.gov (United States)

    Zhang, Si; Huang, Nan; Pan, Xia; Zang, Jing-Lei; Guan, Xin-Xin; Zhang, Jian-Hua; Liu, Liu-Cheng; Lei, Xiao-Yong

    2016-04-25

    Eukaryotic translation initiation factor 4G (eIF4G) is a scaffold component of eukaryotic translation initiation factor 4F (eIF4F) complex, which takes principal part in the initiating of protein synthesis. Both two subtypes (eIF4G1 and eIF4G2) of eIF4G were found to be closely related with various tumors. The eIF4G1 expression is significantly up-regulated in breast cancer, cervical cancer, nasopharyngeal carcinoma, lung squamous cell carcinoma, prostatic carcinoma and other malignant tumors, compared with those in adjacent tissues; and the eIF4G2 is obviously over-expressed in diffuse large B cell lymphoma and acute myeloid leukemia, but low-expressed in bladder transitional cell carcinoma. This paper reviews the progress in the study of the role of eIF4G in tumor genesis, development, diagnosis and prognosis.

  5. Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.

    Science.gov (United States)

    Dunne, O; Weidenhaupt, M; Callow, P; Martel, A; Moulin, M; Perkins, S J; Haertlein, M; Forsyth, V T

    2017-07-01

    Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.

  6. Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Zhang, Hui; Pi, Fengmei; Shu, Dan; Vieweger, Mario; Guo, Peixuan

    2015-01-01

    RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

  7. Eukaryotic diversity in historical soil samples

    NARCIS (Netherlands)

    Moon-van der Staay, S.Y.; Tzeneva, V.A.; Staay, van der G.W.M.; Vos, de W.M.; Smidt, H.; Hackstein, J.H.P.

    2006-01-01

    The eukaryotic biodiversity in historical air-dried samples of Dutch agricultural soil has been assessed by random sequencing of an 18S rRNA gene library and by denaturing gradient gel electrophoresis. Representatives of nearly all taxa of eukaryotic soil microbes could be identified, demonstrating

  8. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    Science.gov (United States)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  9. pHSP70P-EGFP真核表达载体的构建及其在人Chang's肝细胞中的表达%Construction of eukaryotic expression vector pHSP70P-EGFP and its expression in human Chang's liver cell

    Institute of Scientific and Technical Information of China (English)

    高峰; 陈亚军; 杨学文

    2011-01-01

    monoclonal cells were screened by G418 in final concentration of 300 ng/mL. The stimulation of hepatotoxic drug Ketoconazole to human Chang's liver cell induced enhancement of green fluorescence regulated by HSP70 promoter. The relative expression of EGFP mRNA by stimulation of drug in the transfected cells was significantly higher than that of the control group (( = - 14. 21 ,P < 0.05). Conclusion The eukaryotic expression vector of HSP70 promoter and green fluorescent protein was successfully constructed and the monoclonal cells with stable transfection were obtained. The stress expression of HSP70 by stimulation of hepatotoxic drugs was verified. A cell mode for early prediction of hepatotoxic drugs was provided to select new drugs with high-throughput in vitro.

  10. Membrane remodeling and organization: Elements common to prokaryotes and eukaryotes.

    Science.gov (United States)

    Vega-Cabrera, Luz A; Pardo-López, Liliana

    2017-02-01

    Membrane remodeling processes in eukaryotes, such as those involved in endocytosis and intracellular trafficking, are mediated by a large number of structural, accessory and regulatory proteins. These processes occur in all cell types, enabling the exchange of signals and/or nutrients with the external medium and with neighboring cells; likewise, they are required for the intracellular trafficking of various cargo molecules between organelles, as well as the recycling of these structures. Recent studies have demonstrated that some elements of the molecular machinery involved in regulating and mediating endocytosis in eukaryotic cells are also present in some bacteria, where they participate in processes such as cell division, sporulation and signal transduction. However, the mechanism whereby this prokaryotic machinery carries out such functions has barely begun to be elucidated. This review summarizes recent information about the cytoskeletal and membrane-organizing proteins for which bacterial homologs have been identified; given their known functions, they may be considered to be part of an ancestral membrane organization system that first emerged in prokaryotes and which further evolved into the more complex regulatory networks operating in eukaryotes. © 2017 IUBMB Life, 69(2):55-62, 2017.

  11. The ring of life hypothesis for eukaryote origins is supported by multiple kinds of data.

    Science.gov (United States)

    McInerney, James; Pisani, Davide; O'Connell, Mary J

    2015-09-26

    The literature is replete with manuscripts describing the origin of eukaryotic cells. Most of the models for eukaryogenesis are either autogenous (sometimes called slow-drip), or symbiogenic (sometimes called big-bang). In this article, we use large and diverse suites of 'Omics' and other data to make the inference that autogeneous hypotheses are a very poor fit to the data and the origin of eukaryotic cells occurred in a single symbiosis. © 2015 The Author(s).

  12. Organic Based Solar Cells with Morphology Control

    OpenAIRE

    Andersen, Thomas Rieks; Bundgaard, Eva; Jørgensen, Mikkel

    2013-01-01

    The field of organic solar cells has in the last years gone through an impressive development with efficiencies reported up to 12 %. For organic solar cells to take the leap from primarily being a laboratory scale technology to being utilized as renewable energy source, several issues need to be addressed. Among these are a more direct transfer of new materials tested on a laboratory scale to large scale production than offered by spincoating, a method offering direct control of the morpholog...

  13. Translational control in germline stem cell development.

    Science.gov (United States)

    Slaidina, Maija; Lehmann, Ruth

    2014-10-13

    Stem cells give rise to tissues and organs during development and maintain their integrity during adulthood. They have the potential to self-renew or differentiate at each division. To ensure proper organ growth and homeostasis, self-renewal versus differentiation decisions need to be tightly controlled. Systematic genetic studies in Drosophila melanogaster are revealing extensive regulatory networks that control the switch between stem cell self-renewal and differentiation in the germline. These networks, which are based primarily on mutual translational repression, act via interlocked feedback loops to provide robustness to this important fate decision.

  14. Eukaryotic versus prokaryotic marine picoplankton ecology.

    Science.gov (United States)

    Massana, Ramon; Logares, Ramiro

    2013-05-01

    Marine microorganisms contribute markedly to global biomass and ecosystem function. They include a diverse collection of organisms differing in cell size and in evolutionary history. In particular, microbes within the picoplankton are similar in size but belong to two drastically different cellular plans, the prokaryotes and the eukaryotes. Compared with larger organisms, prokaryotes and picoeukaryotes share ecological features, such as high specific activity, large and constant abundances, and high dispersal potential. Still, there are some aspects where their different cell organization influences their ecological performance. First, prokaryotes have a huge metabolic versatility and are involved in all biogeochemical cycles, whereas picoeukaryotes are metabolically less flexible but can exploit diverse predatory life strategies due to their phagocytic capacity. Second, sexual reproduction is absent in prokaryotes but may be present in picoeukaryotes, thus determining different evolutionary diversification dynamics and making species limits clearer in picoeukaryotes. Finally, it is plausible that picoeukaryotes are less flexible to enter a reversible state of low metabolic activity, thus picoeukaryote assemblages may have fewer rare species and may be less resilient to environmental change. In summary, lumping together pico-sized microbes may be convenient for some ecological studies, but it is also important to keep in mind their differences.

  15. Myosin domain evolution and the primary divergence of eukaryotes.

    Science.gov (United States)

    Richards, Thomas A; Cavalier-Smith, Thomas

    2005-08-25

    Eukaryotic cells have two contrasting cytoskeletal and ciliary organizations. The simplest involves a single cilium-bearing centriole, nucleating a cone of individual microtubules (probably ancestral for unikonts: animals, fungi, Choanozoa and Amoebozoa). In contrast, bikonts (plants, chromists and all other protozoa) were ancestrally biciliate with a younger anterior cilium, converted every cell cycle into a dissimilar posterior cilium and multiple ciliary roots of microtubule bands. Here we show by comparative genomic analysis that this fundamental cellular dichotomy also involves different myosin molecular motors. We found 37 different protein domain combinations, often lineage-specific, and many previously unidentified. The sequence phylogeny and taxonomic distribution of myosin domain combinations identified five innovations that strongly support unikont monophyly and the primary bikont/unikont bifurcation. We conclude that the eukaryotic cenancestor (last common ancestor) had a cilium, mitochondria, pseudopodia, and myosins with three contrasting domain combinations and putative functions.

  16. Wnt signaling and stem cell control

    Institute of Scientific and Technical Information of China (English)

    Roel Nusse

    2008-01-01

    Wnt signaling has been implicated in the control over various types of stem cells and may act as a niche factor to maintain stem cells in a self-renewing state.As currently understood,Wnt proteins bind to receptors of the Frizzled and LRP families on the cell surface.Through several cytoplasmic relay components,the signal is transduced to B-catenin,which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes.Wnts can also signal through tyrosine kinase receptors,in particular the ROR and RYK receptors,leading to alternative modes of Wnt signaling.During the growth of tissues,these ligands and receptors are dynamically expressed,often transcriptionally controlled by Wnt signals themselves,to ensure the right balance between proliferation and differentiation.Isolated Wnt proteins are active on a variety of stem cells,including neural,mammary and embryonic stem cells.In general,Wnt proteins act to maintain the undifferentiated state of stem cells,while other growth factors instruct the cells to proliferate.These other factors include FGF and EGF,signaling through tyrosine kinase pathways.

  17. Bacterial proteins pinpoint a single eukaryotic root.

    Science.gov (United States)

    Derelle, Romain; Torruella, Guifré; Klimeš, Vladimír; Brinkmann, Henner; Kim, Eunsoo; Vlček, Čestmír; Lang, B Franz; Eliáš, Marek

    2015-02-17

    The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

  18. Evolution of Copper Transporting ATPases in Eukaryotic Organisms

    OpenAIRE

    Gupta, Arnab; Lutsenko, Svetlana

    2012-01-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases i...

  19. Eukaryotic expression of bovine Neospora caninumMAG1 gene in Vero cells%牛源犬新孢子虫MAG1基因的克隆及在Vero细胞中表达

    Institute of Scientific and Technical Information of China (English)

    焦石; 贾立军; 刘明明; 黄国明; 张守发

    2011-01-01

    为了解牛源犬新孢子虫MAG1基因的生物学特性,本实验应用PCR技术扩增牛源犬新孢子虫MAG1基因,构建真核表达重组质粒pVAX-MAG1,将鉴定正确的pVAX-MAG1重组质粒转染Vero细胞,应用间接荧光检测方法(1FA)和western blot技术检测MAG1基因在Vero细胞中的表达.结果显示,扩增的牛源犬新孢子虫MAG1基因长度为1047bp,与GenBank中登录的MAG1 (EF580924.1)核苷酸序列同源性为99%,IFA检测MAG1基因在Vero细胞中获得瞬时表达,western blot分析表达蛋白的分子量为39 ku,具有较好的反应原性.本实验为新孢子虫病核酸疫苗与诊断试剂盒的研究奠定了基础.%To inestigate the biological characteristics of bovine Neospora caninum MAGI gene, the gene was amplified by PCR and inserted into eukaryotic expression vector. The resultant recombinant plasmid of pVAX-MAG 1 was transfected into Vero cells and the expression of MAGI was detected by indirect immunofluorescent assay, and western blot showed that the molecular weight of the protein was 39 ku and the protein had a positve reaction with anti MAGI serum. The results could be useful to DNA vaccine development.

  20. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

    Science.gov (United States)

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias

    2010-12-01

    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  1. Searching for the role of protein phosphatases in eukaryotic microorganisms

    Directory of Open Access Journals (Sweden)

    da-Silva A.M.

    1999-01-01

    Full Text Available Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively. Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

  2. Complementing the Eukaryotic Protein Interactome.

    Directory of Open Access Journals (Sweden)

    Robert Pesch

    Full Text Available Protein interaction networks are important for the understanding of regulatory mechanisms, for the explanation of experimental data and for the prediction of protein functions. Unfortunately, most interaction data is available only for model organisms. As a possible remedy, the transfer of interactions to organisms of interest is common practice, but it is not clear when interactions can be transferred from one organism to another and, thus, the confidence in the derived interactions is low. Here, we propose to use a rich set of features to train Random Forests in order to score transferred interactions. We evaluated the transfer from a range of eukaryotic organisms to S. cerevisiae using orthologs. Directly transferred interactions to S. cerevisiae are on average only 24% consistent with the current S. cerevisiae interaction network. By using commonly applied filter approaches the transfer precision can be improved, but at the cost of a large decrease in the number of transferred interactions. Our Random Forest approach uses various features derived from both the target and the source network as well as the ortholog annotations to assign confidence values to transferred interactions. Thereby, we could increase the average transfer consistency to 85%, while still transferring almost 70% of all correctly transferable interactions. We tested our approach for the transfer of interactions to other species and showed that our approach outperforms competing methods for the transfer of interactions to species where no experimental knowledge is available. Finally, we applied our predictor to score transferred interactions to 83 targets species and we were able to extend the available interactome of B. taurus, M. musculus and G. gallus with over 40,000 interactions each. Our transferred interaction networks are publicly available via our web interface, which allows to inspect and download transferred interaction sets of different sizes, for various

  3. Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

    Science.gov (United States)

    Yu, Geng; Rosenberg, Julian N; Betenbaugh, Michael J; Oyler, George A

    2015-12-01

    Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring.

  4. Control of apoptosis by asymmetric cell division.

    Directory of Open Access Journals (Sweden)

    Julia Hatzold

    2008-04-01

    Full Text Available Asymmetric cell division and apoptosis (programmed cell death are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well.

  5. Kingdoms Protozoa and Chromista and the eozoan root of the eukaryotic tree.

    Science.gov (United States)

    Cavalier-Smith, Thomas

    2010-06-23

    I discuss eukaryotic deep phylogeny and reclassify the basal eukaryotic kingdom Protozoa and derived kingdom Chromista in the light of multigene trees. I transfer the formerly protozoan Heliozoa and infrakingdoms Alveolata and Rhizaria into Chromista, which is sister to kingdom Plantae and arguably originated by synergistic double internal enslavement of green algal and red algal cells. I establish new subkingdoms (Harosa; Hacrobia) for the expanded Chromista. The protozoan phylum Euglenozoa differs immensely from other eukaryotes in its nuclear genome organization (trans-spliced multicistronic transcripts), mitochondrial DNA organization, cytochrome c-type biogenesis, cell structure and arguably primitive mitochondrial protein-import and nuclear DNA prereplication machineries. The bacteria-like absence of mitochondrial outer-membrane channel Tom40 and DNA replication origin-recognition complexes from trypanosomatid Euglenozoa roots the eukaryotic tree between Euglenozoa and all other eukaryotes (neokaryotes), or within Euglenozoa. Given their unique properties, I segregate Euglenozoa from infrakingdom Excavata (now comprising only phyla Percolozoa, Loukozoa, Metamonada), grouping infrakingdoms Euglenozoa and Excavata as the ancestral protozoan subkingdom Eozoa. I place phylum Apusozoa within the derived protozoan subkingdom Sarcomastigota. Clarifying early eukaryote evolution requires intensive study of properties distinguishing Euglenozoa from neokaryotes and Eozoa from neozoa (eukaryotes except Eozoa; ancestrally defined by haem lyase).

  6. Controlling cell-cell interactions using surface acoustic waves.

    Science.gov (United States)

    Guo, Feng; Li, Peng; French, Jarrod B; Mao, Zhangming; Zhao, Hong; Li, Sixing; Nama, Nitesh; Fick, James R; Benkovic, Stephen J; Huang, Tony Jun

    2015-01-06

    The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.

  7. A high-affinity molybdate transporter in eukaryotes.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  8. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa.

    Science.gov (United States)

    Cavalier-Smith, T

    2002-03-01

    Eukaryotes and archaebacteria form the clade neomura and are sisters, as shown decisively by genes fragmented only in archaebacteria and by many sequence trees. This sisterhood refutes all theories that eukaryotes originated by merging an archaebacterium and an alpha-proteobacterium, which also fail to account for numerous features shared specifically by eukaryotes and actinobacteria. I revise the phagotrophy theory of eukaryote origins by arguing that the essentially autogenous origins of most eukaryotic cell properties (phagotrophy, endomembrane system including peroxisomes, cytoskeleton, nucleus, mitosis and sex) partially overlapped and were synergistic with the symbiogenetic origin of mitochondria from an alpha-proteobacterium. These radical innovations occurred in a derivative of the neomuran common ancestor, which itself had evolved immediately prior to the divergence of eukaryotes and archaebacteria by drastic alterations to its eubacterial ancestor, an actinobacterial posibacterium able to make sterols, by replacing murein peptidoglycan by N-linked glycoproteins and a multitude of other shared neomuran novelties. The conversion of the rigid neomuran wall into a flexible surface coat and the associated origin of phagotrophy were instrumental in the evolution of the endomembrane system, cytoskeleton, nuclear organization and division and sexual life-cycles. Cilia evolved not by symbiogenesis but by autogenous specialization of the cytoskeleton. I argue that the ancestral eukaryote was uniciliate with a single centriole (unikont) and a simple centrosomal cone of microtubules, as in the aerobic amoebozoan zooflagellate Phalansterium. I infer the root of the eukaryote tree at the divergence between opisthokonts (animals, Choanozoa, fungi) with a single posterior cilium and all other eukaryotes, designated 'anterokonts' because of the ancestral presence of an anterior cilium. Anterokonts comprise the Amoebozoa, which may be ancestrally unikont, and a vast

  9. Ustilago maydis Rho1 and 14-3-3 homologues participate in pathways controlling cell separation and cell polarity.

    Science.gov (United States)

    Pham, Cau D; Yu, Zhanyang; Sandrock, Björn; Bölker, Michael; Gold, Scott E; Perlin, Michael H

    2009-07-01

    Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.

  10. Paleobiological Perspectives on Early Eukaryotic Evolution

    Science.gov (United States)

    Knoll, Andrew H.

    2014-01-01

    Eukaryotic organisms radiated in Proterozoic oceans with oxygenated surface waters, but, commonly, anoxia at depth. Exceptionally preserved fossils of red algae favor crown group emergence more than 1200 million years ago, but older (up to 1600–1800 million years) microfossils could record stem group eukaryotes. Major eukaryotic diversification ∼800 million years ago is documented by the increase in the taxonomic richness of complex, organic-walled microfossils, including simple coenocytic and multicellular forms, as well as widespread tests comparable to those of extant testate amoebae and simple foraminiferans and diverse scales comparable to organic and siliceous scales formed today by protists in several clades. Mid-Neoproterozoic establishment or expansion of eukaryophagy provides a possible mechanism for accelerating eukaryotic diversification long after the origin of the domain. Protists continued to diversify along with animals in the more pervasively oxygenated oceans of the Phanerozoic Eon. PMID:24384569

  11. Crystal structure of the eukaryotic ribosome.

    Science.gov (United States)

    Ben-Shem, Adam; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2010-11-26

    Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

  12. Eukaryotic diversity in historical soil samples.

    Science.gov (United States)

    Moon-van der Staay, Seung Yeo; Tzeneva, Vesela A; van der Staay, Georg W M; de Vos, Willem M; Smidt, Hauke; Hackstein, Johannes H P

    2006-09-01

    The eukaryotic biodiversity in historical air-dried samples of Dutch agricultural soil has been assessed by random sequencing of an 18S rRNA gene library and by denaturing gradient gel electrophoresis. Representatives of nearly all taxa of eukaryotic soil microbes could be identified, demonstrating that it is possible to study eukaryotic microbiota in samples from soil archives that have been stored for more than 30 years at room temperature. In a pilot study, 41 sequences were retrieved that could be assigned to fungi and a variety of aerobic and anaerobic protists such as cercozoans, ciliates, xanthophytes (stramenopiles), heteroloboseans, and amoebozoans. A PCR-denaturing gradient gel electrophoresis analysis of samples collected between 1950 and 1975 revealed significant changes in the composition of the eukaryotic microbiota.

  13. Reciprocal control of cell proliferation and migration

    Directory of Open Access Journals (Sweden)

    De Donatis Alina

    2010-09-01

    Full Text Available Abstract In adult tissue the quiescent state of a single cell is maintained by the steady state conditions of its own microenvironment for what concern both cell-cell as well as cell-ECM interaction and soluble factors concentration. Physiological or pathological conditions can alter this quiescent state through an imbalance of both soluble and insoluble factors that can trigger a cellular phenotypic response. The kind of cellular response depends by many factors but one of the most important is the concentration of soluble cytokines sensed by the target cell. In addition, due to the intrinsic plasticity of many cellular types, every single cell is able, in response to the same stimulus, to rapidly switch phenotype supporting minimal changes of microenviromental cytokines concentration. Wound healing is a typical condition in which epithelial, endothelial as well as mesenchymal cells are firstly subjected to activation of their motility in order to repopulate the damaged region and then they show a strong proliferative response in order to successfully complete the wound repair process. This schema constitute the leitmotif of many other physiological or pathological conditions such as development vasculogenesis/angiogenesis as well as cancer outgrowth and metastasis. Our review focuses on the molecular mechanisms that control the starting and, eventually, the switching of cellular phenotypic outcome in response to changes in the symmetry of the extracellular environment.

  14. Eukaryote-to-eukaryote gene transfer gives rise to genome mosaicism in euglenids

    Directory of Open Access Journals (Sweden)

    Weber Andreas PM

    2011-04-01

    Full Text Available Abstract Background Euglenophytes are a group of photosynthetic flagellates possessing a plastid derived from a green algal endosymbiont, which was incorporated into an ancestral host cell via secondary endosymbiosis. However, the impact of endosymbiosis on the euglenophyte nuclear genome is not fully understood due to its complex nature as a 'hybrid' of a non-photosynthetic host cell and a secondary endosymbiont. Results We analyzed an EST dataset of the model euglenophyte Euglena gracilis using a gene mining program designed to detect laterally transferred genes. We found E. gracilis genes showing affinity not only with green algae, from which the secondary plastid in euglenophytes evolved, but also red algae and/or secondary algae containing red algal-derived plastids. Phylogenetic analyses of these 'red lineage' genes suggest that E. gracilis acquired at least 14 genes via eukaryote-to-eukaryote lateral gene transfer from algal sources other than the green algal endosymbiont that gave rise to its current plastid. We constructed an EST library of the aplastidic euglenid Peranema trichophorum, which is a eukaryovorous relative of euglenophytes, and also identified 'red lineage' genes in its genome. Conclusions Our data show genome mosaicism in E. gracilis and P. trichophorum. One possible explanation for the presence of these genes in these organisms is that some or all of them were independently acquired by lateral gene transfer and contributed to the successful integration and functioning of the green algal endosymbiont as a secondary plastid. Alternative hypotheses include the presence of a phagocytosed alga as the single source of those genes, or a cryptic tertiary endosymbiont harboring secondary plastid of red algal origin, which the eukaryovorous ancestor of euglenophytes had acquired prior to the secondary endosymbiosis of a green alga.

  15. Evolution of copper transporting ATPases in eukaryotic organisms.

    Science.gov (United States)

    Gupta, Arnab; Lutsenko, Svetlana

    2012-04-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution.

  16. pcDNA3.1(+)GDNF真核表达载体的构建及其在真核细胞中的表达%Construction of pcDNA3.1(+) glial cell line-derived neurotrophic factor(GDNF) vector and its expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    赵永波; 张莹; 王枫; 王乔树; 王维治; 李钰; 张贵寅

    2001-01-01

    Objective To construct pcDNA3.1(+)GDNF recombinant eukaryoticexpression plasmid and to investigate its expression in eukaryotic cells. Methods The coding sequence of GDNF was amplified from rat astrocytes by reverse transcription PCR (RT-PCR) and cloned into pcDNA3.1(+) eukaryotic expression vector. The recombinant pcDNA3.1(+)GDNF plasmid was then transfected into eukaryotic cells mediated by using Fu Gene 6 method. Analysis by restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid. GDNF protein and its activity were then determined using pcDNA3.1(+)GDNF plasmid transfected eukaryotic cells. Results RT-PCR product is 640 bp specific segment. Analysis by restricting enzyme digestion and DNA sequencing of pcDNA3.1(+)GDNF recombinant showed results from restricting enzyme were 640 bp and 300 bp segments respectively. DNA sequencing revealed that GDNF cloning was successful. The recombinant plasmid can express active GDNF protein in eukaryotic cells. Conclusion The study on the role of both GDNF and gene therapy is significant in the treatment of Parkinson disease.%目的 构建pcDNA3.1(+)胶质细胞源性神经营养因子(GDNF)真核表达质粒并了解其在真核细胞内的表达。方法 将GDNF逆转录聚合酶链式反应(RT-PCR)产物克隆至pcDNA3.1(+)真核表达载体上,经酶切鉴定及测序分析并以FuGene6介导法转染真核细胞,了解其在细胞内的表达及其表达蛋白的生物学活性。结果 RT-PCR产物为640bp特异片段,pcDNA3.1(+)GDNF重组体经酶切后分别出现640bp和300bp片段,测序分析与文献报道结果完全一致,表明重组pcDNA3.1(+)GDNF表达质粒克隆成功。可见pcDNA3.1(+)GDNF质粒在真核动物细胞中得到表达,GDNF蛋白能够刺激含多巴胺的细胞生长,表明重组质粒能在真核动物细胞中表达出具有活性的GDNF蛋白。结论 以FuGene6介导pcDNA3.1(+)GDNF质粒转染真核细胞为基因治疗

  17. Fluidic control over cell proliferation and chemotaxis

    Science.gov (United States)

    Groisman, Alex

    2006-03-01

    Microscopic flows are almost always stable and laminar that allows precise control of chemical environment in micro-channels. We describe design and operation of several microfluidic devices, in which various types of environments are created for different experimental assays with live cells. In a microfluidic chemostat, colonies of non-adherent bacterial and yeast cells are trapped in micro-chambers with walls permeable for chemicals. Fast chemical exchange between the chambers and nearby flow-through channels creates essentially chemostatic medium conditions in the chambers and leads to exponential growth of the colonies up to very high cell densities. Another microfluidic device allows creation of linear concentration profiles of a pheromone (α-factor) across channels with non-adherent yeast cells, without exposure of the cells to flow or other mechanical perturbation. The concentration profile remains stable for hours enabling studies of chemotropic response of the cells to the pheromone gradient. A third type of the microfluidic devices is used to study chemotaxis of human neutrophils exposed to gradients of a chemoattractant (fMLP). The devices generate concentration profiles of various shapes, with adjustable steepness and mean concentration. The ``gradient'' of the chemoattractant can be imposed and reversed within less than a second, allowing repeated quantitative experiments.

  18. CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

  19. Kingdoms Protozoa and Chromista and the eozoan root of the eukaryotic tree

    OpenAIRE

    CAVALIER-SMITH, THOMAS

    2009-01-01

    I discuss eukaryotic deep phylogeny and reclassify the basal eukaryotic kingdom Protozoa and derived kingdom Chromista in the light of multigene trees. I transfer the formerly protozoan Heliozoa and infrakingdoms Alveolata and Rhizaria into Chromista, which is sister to kingdom Plantae and arguably originated by synergistic double internal enslavement of green algal and red algal cells. I establish new subkingdoms (Harosa; Hacrobia) for the expanded Chromista. The protozoan phylum Euglenozoa ...

  20. Controlling Functional Group Architecture in Artificial Cells

    Science.gov (United States)

    2015-07-02

    further enable enzyme encapsulation to improve the efficiency of light-driven hydrogen fuel production. 5. Changes in key personnel, if applicable : -None ...Controlling Functional Group Architecture in Artificial Cells 5a. CONTRACT NUMBER W9132T-14-2-0002 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6...cycloadditions to modify reactive groups within the phospholipid membrane structure and how the nature of the reactive elements, the copper catalyst

  1. Carbon nanotubes for stem cell control

    Directory of Open Access Journals (Sweden)

    David A. Stout

    2012-07-01

    Full Text Available In the past decade, two major advancements have transformed the world of tissue engineering and regenerative medicine—stem cells and carbon nano-dimensional materials. In the past, stem cell therapy seemed like it may present a cure for all medical ailments, but problems arose (i.e., immune system clearance, control of differentiation in the body, etc. that have hindered progress. But, with the synergy of carbon nano-dimensional materials, researchers have been able to overcome these tissue engineering and regenerative medicine obstacles and have begun developing treatments for strokes, bone failure, cardiovascular disease, and many other conditions. Here, we briefly review research involving carbon nanotubes which are relevant to the tissue engineering and regenerative medicine field with a special emphasis on carbon nanotube applications for stem cell delivery, drug delivery applications, and their use as improved medical devices.

  2. Eu-Detect: An algorithm for detecting eukaryotic sequences in metagenomic data sets

    Indian Academy of Sciences (India)

    Monzoorul Haque Mohammed; Sudha Chadaram Dinakar; Dinakar Komanduri; Tarini Shankar Ghosh; Sharmila S Mande

    2011-09-01

    Physical partitioning techniques are routinely employed (during sample preparation stage) for segregating the prokaryotic and eukaryotic fractions of metagenomic samples. In spite of these efforts, several metagenomic studies focusing on bacterial and archaeal populations have reported the presence of contaminating eukaryotic sequences inmetagenomic data sets. Contaminating sequences originate not only from genomes of micro-eukaryotic species but also from genomes of (higher) eukaryotic host cells. The latter scenario usually occurs in the case of host-associatedmetagenomes. Identification and removal of contaminating sequences is important, since these sequences not only impact estimates of microbial diversity but also affect the accuracy of several downstream analyses. Currently, the computational techniques used for identifying contaminating eukaryotic sequences, being alignment based, are slow, inefficient, and require huge computing resources. In this article, we present Eu-Detect, an alignment-free algorithm that can rapidly identify eukaryotic sequences contaminating metagenomic data sets. Validation results indicate that on a desktop with modest hardware specifications, the Eu-Detect algorithm is able to rapidly segregate DNA sequence fragments of prokaryotic and eukaryotic origin, with high sensitivity. A Web server for the Eu-Detect algorithm is available at http://metagenomics.atc.tcs.com/Eu-Detect/.

  3. Horizontal transfer of bacterial polyphosphate kinases to eukaryotes: implications for the ice age and land colonisation.

    Science.gov (United States)

    Whitehead, Michael P; Hooley, Paul; W Brown, Michael R

    2013-06-05

    Studies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes. On - line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species

  4. Eukaryotes dominate new production in the Sargasso Sea

    Science.gov (United States)

    Fawcett, S. E.; Lomas, M. W.; Ward, B. B.; Casey, J. R.; Sigman, D. M.

    2010-12-01

    The vast subtropical ocean gyres are considered unproductive “deserts” due to the extremely low concentrations of essential nutrients in their sunlit surface waters. Because of intense upper ocean stratification, phytoplankton growth in the subtropical gyres is limited by the slow supply of nitrate from below, and is assumed to be supported predominantly by “regenerated” nitrogen (N): ammonium and other reduced N sources recycled in surface waters. The phytoplankton assemblage of the subtropical Sargasso Sea is dominated by the prokaryotic cyanobacteria, Prochlorococcus and Synechococcus, which occur in very high cell numbers compared to the rarer, and usually larger, eukaryotic algae. Coupling flow cytometry and a new high-sensitivity method for N isotope analysis, we measure the 15N/14N of major phytoplankton taxa and other biologically distinct particle populations collected from the surface waters of the Sargasso Sea during the stratified summer period. We find that the cyanobacteria and eukaryotic phytoplankton show distinct N isotope signatures, indicating that they utilize different sources of N for growth. Prochlorococcus and Synechococcus have a uniformly low 15N/14N, consistent with the expectation that these phytoplankton rely on regenerated N. However, the 15N/14N of eukaryotic phytoplankton is higher and more variable, with a mean 15N/14N comparable to the new nitrate supply from below, indicating that eukaryotes dominate the consumption of this nitrate and rely on it for more than half of their N requirement. Using our measured 15N/14N values for the various sorted autotrophic populations, we calculate eukaryote-specific summer f-ratios of 0.6-0.67 and total community summer f-ratios of 0.15-0.23. These values are higher than those based on comparison of primary production and sediment-trap derived organic carbon (C) export, and agree well with annual f-ratio estimates implied by geochemical tracers. The high 15N/14N of eukaryotic biomass can

  5. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

    Science.gov (United States)

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-07-14

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

  6. The eukaryotic ancestor had a complex ubiquitin signaling system of archaeal origin.

    Science.gov (United States)

    Grau-Bové, Xavier; Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2015-03-01

    The origin of the eukaryotic cell is one of the most important transitions in the history of life. However, the emergence and early evolution of eukaryotes remains poorly understood. Recent data have shown that the last eukaryotic common ancestor (LECA) was much more complex than previously thought. The LECA already had the genetic machinery encoding the endomembrane apparatus, spliceosome, nuclear pore, and myosin and kinesin cytoskeletal motors. It is unclear, however, when the functional regulation of these cellular components evolved. Here, we address this question by analyzing the origin and evolution of the ubiquitin (Ub) signaling system, one of the most important regulatory layers in eukaryotes. We delineated the evolution of the whole Ub, Small-Ub-related MOdifier (SUMO), and Ub-fold modifier 1 (Ufm1) signaling networks by analyzing representatives from all major eukaryotic, bacterial, and archaeal lineages. We found that the Ub toolkit had a pre-eukaryotic origin and is present in three extant archaeal groups. The pre-eukaryotic Ub toolkit greatly expanded during eukaryogenesis, through massive gene innovation and diversification of protein domain architectures. This resulted in a LECA with essentially all of the Ub-related genes, including the SUMO and Ufm1 Ub-like systems. Ub and SUMO signaling further expanded during eukaryotic evolution, especially labeling and delabeling enzymes responsible for substrate selection. Additionally, we analyzed protein domain architecture evolution and found that multicellular lineages have the most complex Ub systems in terms of domain architectures. Together, we demonstrate that the Ub system predates the origin of eukaryotes and that a burst of innovation during eukaryogenesis led to a LECA with complex posttranslational regulation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Construction of eukaryotic expression vector of Der p2 gene and its expression in mouse dendritic cells%Der p 2基因真核表达载体构建及其在小鼠树突状细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    毕玉田; 王彦; 吴奎; 王长征; 钱桂生

    2008-01-01

    Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA.METHODS: Mouse bone marrow-derived dendritic cells were in vitro isolated and cultured.Complete Der p 2 cDNA was spliced from prokaryotic expression vector plambd-Der p 2, and then cloned into eukaryotic expression vector pCI-neo (pCI-neo-Der p 2).The positive recombinants pCl-neo-Der p 2 transfected into dendritic cells.Non-transfected and blank vector pCI-neo-transfected dendritic cells were used as controls. MAIN OUTCOME MEASURES: ①Identification of pCI-neo-Der p 2 recombinant plasmid.②Detection of Der p 2 mRNA and protein expression by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot techniques. RESULTS: Sequencing results showed Der p 2 cDNA in pCI-neo-Der p 2 was in coincidence with the sequence registrated in Gene Bank.RT-PCR and Western Blot results showed that expression of Der p 2 mRNA and protein could be detectable in the pCI-neo-Der p 2-transfected dendritic cells. CONCLUSION: The Der p 2 cDNA was successfully constructed into the eukaryotic expression vector, and Der p 2 gene and protein could be expressed efficiently in dendritic cells.

  8. Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lykidis, Athanasios

    2006-12-01

    Phospholipid biosynthetic enzymes produce diverse molecular structures and are often present in multiple forms encoded by different genes. This work utilizes comparative genomics and phylogenetics for exploring the distribution, structure and evolution of phospholipid biosynthetic genes and pathways in 26 eukaryotic genomes. Although the basic structure of the pathways was formed early in eukaryotic evolution, the emerging picture indicates that individual enzyme families followed unique evolutionary courses. For example, choline and ethanolamine kinases and cytidylyltransferases emerged in ancestral eukaryotes, whereas, multiple forms of the corresponding phosphatidyltransferases evolved mainly in a lineage specific manner. Furthermore, several unicellular eukaryotes maintain bacterial-type enzymes and reactions for the synthesis of phosphatidylglycerol and cardiolipin. Also, base-exchange phosphatidylserine synthases are widespread and ancestral enzymes. The multiplicity of phospholipid biosynthetic enzymes has been largely generated by gene expansion in a lineage specific manner. Thus, these observations suggest that phospholipid biosynthesis has been an actively evolving system. Finally, comparative genomic analysis indicates the existence of novel phosphatidyltransferases and provides a candidate for the uncharacterized eukaryotic phosphatidylglycerol phosphate phosphatase.

  9. Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes.

    Science.gov (United States)

    Kohli, Gurjeet S; John, Uwe; Van Dolah, Frances M; Murray, Shauna A

    2016-08-01

    Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success.

  10. From damage response to action potentials: early evolution of neural and contractile modules in stem eukaryotes.

    Science.gov (United States)

    Brunet, Thibaut; Arendt, Detlev

    2016-01-05

    Eukaryotic cells convert external stimuli into membrane depolarization, which in turn triggers effector responses such as secretion and contraction. Here, we put forward an evolutionary hypothesis for the origin of the depolarization-contraction-secretion (DCS) coupling, the functional core of animal neuromuscular circuits. We propose that DCS coupling evolved in unicellular stem eukaryotes as part of an 'emergency response' to calcium influx upon membrane rupture. We detail how this initial response was subsequently modified into an ancient mechanosensory-effector arc, present in the last eukaryotic common ancestor, which enabled contractile amoeboid movement that is widespread in extant eukaryotes. Elaborating on calcium-triggered membrane depolarization, we reason that the first action potentials evolved alongside the membrane of sensory-motile cilia, with the first voltage-sensitive sodium/calcium channels (Nav/Cav) enabling a fast and coordinated response of the entire cilium to mechanosensory stimuli. From the cilium, action potentials then spread across the entire cell, enabling global cellular responses such as concerted contraction in several independent eukaryote lineages. In animals, this process led to the invention of mechanosensory contractile cells. These gave rise to mechanosensory receptor cells, neurons and muscle cells by division of labour and can be regarded as the founder cell type of the nervous system. © 2015 The Authors.

  11. Cloning and expression of Zaire Ebola virus glycoprotein in prokaryotic and eukaryotic cells%扎伊尔埃博拉病毒糖蛋白基因的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    张振清; 张爽; 黄弋; 张波; 胡晓敏; 袁志明

    2014-01-01

    埃博拉病毒属丝状病毒科,能引发动物和人出血热症状,人感染后病死率高达90%以上,目前还没有有效预防和治疗的药物和疫苗。近年来,这种烈性传染病病毒传入我国的可能性不断加大,给我国公共卫生应急体系带来新的挑战。本研究针对埃博拉病毒的最主要结构蛋白———糖蛋白(GP),构建了重组原核表达载体pET28a(+)‐GP1(33~313 aa)、pET28a(+)‐GP1(190~313 aa)、pET28a(+)‐GP2(502~632 aa)、pET28a(+)‐sGP ,以及重组真核表达载体pcDNA3.1(+)‐edited GP、pcDNA3.1(+)‐GP1、pcDNA3.1(+)‐GP。结果表明,GP1(33~313 aa)、GP1(190~313 aa)和sGP能在大肠埃希菌BL21(DE3)中以包涵体的形式表达,GP、GP1和GP2能在HEK293T细胞中表达,但均不能在BHK21细胞中表达。本研究为进一步探索埃博拉病毒GP的结构和功能及GP抗体制备奠定了基础。%Ebola virus belongs to filoviridae .Zaire Ebola virus has a high mortality rate up to 90% .It is transmitted through blood , secretions , organs or other body fluids of infected people or animals . Unfortunately ,there are still no approved drugs and vaccines for Ebola virus infection .Considering the increasing commercial and personal communication with countries in Africa ,the possibility of Ebola virus invading into China by infected animals and human highlights the necessity for the development of effective vaccines .In this study ,a series of glycoproteins (GPs) were expressed by using pET28a(+ ) and pcDNA3 .1 (+ ) in prokaryotic and eukaryotic cells ,respectively .The results showed that GP1(33‐313 aa) ,GP1(190‐313 aa) and nonstructural secretory glycoprotein (sGP) were expressed in Escherichia coli BL21 (DE3)in inclusion bodies ,while no GP2 (502‐632 aa) expression could be detected under the same condition .The edited GP ,GP1 and GP2 were expressed in HEK293T cells

  12. Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

    Science.gov (United States)

    Bidnenko, Vladimir; Nicolas, Pierre; Grylak-Mielnicka, Aleksandra; Delumeau, Olivier; Auger, Sandrine; Aucouturier, Anne; Guerin, Cyprien; Repoila, Francis; Bardowski, Jacek; Aymerich, Stéphane; Bidnenko, Elena

    2017-07-01

    In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

  13. Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Vladimir Bidnenko

    2017-07-01

    Full Text Available In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

  14. A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples.

    Science.gov (United States)

    Dollive, Serena; Peterfreund, Gregory L; Sherrill-Mix, Scott; Bittinger, Kyle; Sinha, Rohini; Hoffmann, Christian; Nabel, Christopher S; Hill, David A; Artis, David; Bachman, Michael A; Custers-Allen, Rebecca; Grunberg, Stephanie; Wu, Gary D; Lewis, James D; Bushman, Frederic D

    2012-07-03

    Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

  15. Organic Based Solar Cells with Morphology Control

    DEFF Research Database (Denmark)

    Andersen, Thomas Rieks

    The field of organic solar cells has in the last years gone through an impressive development with efficiencies reported up to 12 %. For organic solar cells to take the leap from primarily being a laboratory scale technology to being utilized as renewable energy source, several issues need...... to be addressed. Among these are a more direct transfer of new materials tested on a laboratory scale to large scale production than offered by spincoating, a method offering direct control of the morphology in the active layer, and a more environmental friendly processing, where the vast use of organic solvents...... offers a great challenge. In this thesis the development of inks with a pre-arranged morphology was attempted by two methods. First by grafting of silicon nanoparticles with an organic phenylene vinylene oligomer, the resulting particles were analyzed by 1H-NMR, absorption spectroscopy, Atomic Force...

  16. Control of division and differentiation of plant stem cells and their derivatives.

    Science.gov (United States)

    Nieuwland, Jeroen; Scofield, Simon; Murray, James A H

    2009-12-01

    The core mechanism of the plant cell cycle is conserved with all other eukaryotes but several aspects are unique to plant cells. Key characteristics of plant development include indeterminate growth and repetitive organogenesis derived from stem cell pools and they may explain the existence of the high number of cell cycle regulators in plants. In this review, we give an overview of the plant cell cycle and its regulatory components. Furthermore, we discuss the cell cycle aspects of plant stem cell maintenance and how the cell cycle relates to cellular differentiation during development. We exemplify this transition by focusing on organ initiation in the shoot.

  17. Oceanographic structure drives the assembly processes of microbial eukaryotic communities.

    Science.gov (United States)

    Monier, Adam; Comte, Jérôme; Babin, Marcel; Forest, Alexandre; Matsuoka, Atsushi; Lovejoy, Connie

    2015-03-17

    Arctic Ocean microbial eukaryote phytoplankton form subsurface chlorophyll maximum (SCM), where much of the annual summer production occurs. This SCM is particularly persistent in the Western Arctic Ocean, which is strongly salinity stratified. The recent loss of multiyear sea ice and increased particulate-rich river discharge in the Arctic Ocean results in a greater volume of fresher water that may displace nutrient-rich saltier waters to deeper depths and decrease light penetration in areas affected by river discharge. Here, we surveyed microbial eukaryotic assemblages in the surface waters, and within and below the SCM. In most samples, we detected the pronounced SCM that usually occurs at the interface of the upper mixed layer and Pacific Summer Water (PSW). Poorly developed SCM was seen under two conditions, one above PSW and associated with a downwelling eddy, and the second in a region influenced by the Mackenzie River plume. Four phylogenetically distinct communities were identified: surface, pronounced SCM, weak SCM and a deeper community just below the SCM. Distance-decay relationships and phylogenetic structure suggested distinct ecological processes operating within these communities. In the pronounced SCM, picophytoplanktons were prevalent and community assembly was attributed to water mass history. In contrast, environmental filtering impacted the composition of the weak SCM communities, where heterotrophic Picozoa were more numerous. These results imply that displacement of Pacific waters to greater depth and increased terrigenous input may act as a control on SCM development and result in lower net summer primary production with a more heterotroph dominated eukaryotic microbial community.

  18. Non-coding RNAs: the architects of eukaryotic complexity.

    Science.gov (United States)

    Mattick, J S

    2001-11-01

    Around 98% of all transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.

  19. Gram-Negative Bacterial Sensors for Eukaryotic Signal Molecules

    Directory of Open Access Journals (Sweden)

    Olivier Lesouhaitier

    2009-09-01

    Full Text Available Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction

  20. Epigenetic control of cell identity and plasticity

    KAUST Repository

    Orlando, Valerio

    2014-04-02

    The DNA centered dogma for genetic information and cell identity is now evolving into a much more complex and flexible dimension provided by the discovery of the Epigenome. This comprises those chromosome structural and topological components that complement DNA information and contribute to genome functional organization. Current concept is that the Epigenome constitutes the dynamic molecular interface allowing the Genome to interact with the Environment. Exploring how the genome interacts with the environment is a key to fully understand cellular and complex organism mechanisms of adaptation and plasticity. Our work focuses on the role of an essential, specialized group or chromatin associated proteins named Polycomb (PcG) that control maintenance of transcription programs during development and in adult life. In particular PcG proteins exert epigenetic “memory” function by modifying chromosome structures at various levels to maintain gene silencing in particular through cell division. While in the past decade substantial progress was made in understanding PcG mechanisms acting in development and partially during cell cycle, very little is known about their role in adult post-mitotic tissues and more in general the role of the epigenome in adaptation. To this, we studied the role of PcG in the context of mammalian skeletal muscle cell differentiation. We previously reported specific dynamics of PRC2 proteins in myoblasts and myotubes, in particular the dynamics of PcG Histone H3 K27 Methyl Transferases (HMT), EZH2 and EZH1, the latter apparently replacing for EZH2 in differentiated myotubes. Ezh1 protein, although almost identical to Ezh2, shows a weak H3K27 HMT activity and its primary function remains elusive. Recent ChIPseq studies performed in differentiating muscle cells revealed that Ezh1 associates with active and not repressed regulatory regions to control RNA pol II elongation. Since H3K27 tri-methylation levels are virtually steady in non

  1. Small RNA Control of Cell-to-Cell Communication in Vibrio Harveyi and Vibrio Cholerae

    Science.gov (United States)

    Svenningsen, Sine Lo

    Quorum sensing is a process of cell-to-cell communication, by which bacteria coordinate gene expression and behavior on a population-wide scale. Quorum sensing is accomplished through production, secretion, and subsequent detection of chemical signaling molecules termed autoinducers. The human pathogen Vibrio cholerae and the marine bioluminescent bacterium Vibrio harveyi incorporate information from multiple autoinducers, and also environmental signals and metabolic cues into their quorum-sensing pathways. At the core of these pathways lie several homologous small regulatory RNA molecules, the Quorum Regulatory RNAs. Small noncoding RNAs have emerged throughout the bacterial and eukaryotic kingdoms as key regulators of behavioral and developmental processes. Here, I review our present understanding of the role of the Qrr small RNAs in integrating quorum-sensing signals and in regulating the individual cells response to this information.

  2. P27 in cell cycle control and cancer

    DEFF Research Database (Denmark)

    Møller, Michael Boe

    2000-01-01

    In order to survive, cells need tight control of cell cycle progression. The control mechanisms are often lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDKs). The CDK inhibitors (CKIs) are important regulators of the CDKs. As the name implies, CKIs were...

  3. Cell-cycle control by protein kinase B

    NARCIS (Netherlands)

    Kops, G.J.P.L.

    2002-01-01

    Numerous cells in the body divide, and do so in a well-controlled manner. In some situations where this control is deregulated, cells may divide continuously. Such uncontrolled proliferation of cells is thought to be responsible for the onset of cancer. In order for a cell to divide in a normal set

  4. Construction of eukaryotic expression plasmid carrying HSV-TK gene and its expression in HepG2 cells%含HSV-TK基因的真核表达载体的构建及其在HepG2细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    张阳德; 孙颖

    2003-01-01

    Objective:To construct a eukaryotic expression plasmid carrying the HSV-TK gene driven by AFP enhancer and CMV promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC).Methods:The minimal essential DNA fragment of AFP gene enhancer was amplified through PCR from genome DNA of HepG2 cells and cloned into the BglII site of plasmid pcDNA3.1-LUC to construct the recombinant plasmid pAFP-LUC. Then the full length cDNA of HSV-TK was cloned into EcoRI site of the recombinant plasmid pAFP-LUC instead of the Luciferase gene to construct pAFP-TK. The recombinant plasmid pAFP-LUC was transferred into AFP-producing hepatoma cell line (HepG2) and non-AFP-producing nonhepatoma cell line (HeLa) by means of lipofectamine. The expression of Luciferase was tested by Luciferase Assay. Results: The length and sequence of AFP enhancer amplified by PCR were confirmed by agarose gel electrophoresis and DNA sequencing.The length, position and orientation of inserted AFP enhancer in pAFP-LUC were all confirmed correct by the methods of restriction digestion and PCR. And it was confirmed by electrophoresis after restriction digestion that the full length HSV-TK was directedly and successfully cloned into the eukaryotic vector . The transcription of Luciferase gene was under the control of AFP enhancer. The expression of Luciferase gene was detected in HepG2 and HeLa cells. The expression of Luciferase is more potent in HepG2 than in HeLa (P<0.05).Conclusions:Construction of a eukaryotic expression plasmid carrying HSV-TK gene driven by AFP enhancer and CMV promotor and its specific expression in HepG2 cells provide a sound basis for targeted gene therapy for HCC.%目的构建AFP增强子CMV启动子调控下的HSV-TK 真核表达质粒用于肝细胞癌的靶向基因治疗.方法采用PCR方法从HepG2细胞基因组DNA中扩增AFP基因增强子最小的功能片断,插入pcDNA3.1-LUC质粒的Bgl II位点,从而构建重组表达质粒pAFP-LUC.HSV-TK cDNA

  5. Hypusine formation in eukaryotic initiation factor 4D is not reversed when rates or specificity of protein synthesis is altered.

    Science.gov (United States)

    Gordon, E D; Mora, R; Meredith, S C; Lindquist, S L

    1987-12-05

    In mammalian cells, a single major cellular protein (eukaryotic initiation factor 4D) is post-translationally modified by the conversion of a lysine residue into the unusual amino acid hypusine. This modification was reported to occur during mitogen-stimulated growth of lymphocytes but not during quiescence, suggesting that alternative forms of eukaryotic initiation factor 4D might play a role in the regulation of cell growth perhaps through the control of protein synthesis itself (Cooper, H. L., Park, M. H., and Folk, J. E. (1982) Cell 29, 791-797). We took advantage of the drastic changes in translational specificity which occur in heat-shocked cells of Drosophila melanogaster, and of the wide variations in translation rates which occur in response to alterations of growth media in the fungus Saccharomyces cerevisiae, to investigate the relationship between the intracellular level and state of modification of the hypusine-containing protein and the rate and specificity of translation. We also studied whether the hypusine residue in this protein might be subject to further modification or reversion to lysine. Under all conditions examined, the protein was remarkably long-lived. Furthermore, the hypusine persists in this protein as hypusine, without further modification or reversion to lysine. Thus, we observe no correlation between the state of cellular translation and the persistence or reversal of this protein's modification. In addition, the data imply that neither are the state of such key cellular processes as DNA replication, RNA transcription, or carbohydrate metabolism so correlated.

  6. The effect of negative autoregulation on eukaryotic gene expression

    Science.gov (United States)

    Nevozhay, Dmitry; Adams, Rhys; Murphy, Kevin; Josic, Kresimir; Balázsi, G. Ábor

    2009-03-01

    Negative autoregulation is a frequent motif in gene regulatory networks, which has been studied extensively in prokaryotes. Nevertheless, some effects of negative feedback on gene expression in eukaryotic transcriptional networks remain unknown. We studied how the strength of negative feedback regulation affects the characteristics of gene expression in yeast cells carrying synthetic transcriptional cascades. We observed a drastic reduction of gene expression noise and a change in the shape of the dose-response curve. We explained these experimentally observed effects by stochastic simulations and a simple set of algebraic equations.

  7. Evidence for a Minimal Eukaryotic Phosphoproteome?

    NARCIS (Netherlands)

    Diks, Sander H.; Parikh, Kaushal; van der Sijde, Marijke; Joore, Jos; Ritsema, Tita; Peppelenbosch, Maikel P.

    2007-01-01

    Background. Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes. Methodology/Principal Findings. We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in c

  8. Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

    Science.gov (United States)

    Huberman, Eliezer; Baccam, Mekhine J.

    2007-02-27

    The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

  9. Cell biology of prokaryotic organelles.

    Science.gov (United States)

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-10-01

    Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.

  10. Cell Division, a new open access online forum for and from the cell cycle community

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2006-04-01

    Full Text Available Abstract Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.

  11. Structure and Control Strategies of Fuel Cell Vehicle

    Institute of Scientific and Technical Information of China (English)

    宋建国; 张承宁; 孙逢春; 钟秋海

    2004-01-01

    The structure and kinds of the fuel cell vehicle (FCV) and the mathematical model of the fuel cell processor are discussed in detail. FCV includes many parts: the fuel cell thermal and water management, fuel supply, air supply and distribution, AC motor drive, main and auxiliary power management, and overall vehicle control system. So it requires different kinds of control strategies, such as the PID method, zero-pole method, optimal control method, fuzzy control and neural network control. Along with the progress of control method, the fuel cell vehicle's stability and reliability is up-and-up. Experiment results show FCV has high energy efficiency.

  12. Towards a palaeoecological model of the Mesoproterozoic Taoudeni Basin, Mauritania, Northwestern Africa: implications for early eukaryote evolution

    Science.gov (United States)

    Beghin, Jérémie; Guilbaud, Romain; Poulton, Simon W.; Gueneli, Nur; Brocks, Jochen J.; Storme, Jean-Yves; Blanpied, Christian; Javaux, Emmanuelle J.

    2016-04-01

    The mid-Proterozoic rock record preserves a relatively moderate diversity of early eukaryotes, despite the early evolution of fundamental features of the eukaryotic cell. Common hypotheses involve the redox state of stratified oceans with oxic shallow waters, euxinic mid-depth waters, and anoxic and ferruginous deep waters during this time period. Mid-Proterozoic eukaryotes would have found suitable ecological niches in estuarine, fluvio-deltaic and coastal shallow marine environments near nutrient sources, while N2-fixing photoautotrophs bacteria would have been better competitors than eukaryotic algae in nutrient-poor niches. Here, we present the first palaeoecological model of the late Mesoproterozoic Taoudeni Basin, Mauritania, Northwestern Africa. Previous palaeontological studies in the basin reported stromatolites, a low diversity of microfossils - including one species of presumed eukaryotes: verrucae-bearing acritarch - and biomarkers of anoxygenic phototrophic purple and green sulfur bacteria, cyanobacteria and microaerophilic methanotrophs. However, no biomarkers diagnostic for crown group eukaryotes were reported so far. In addition to exceptionally well preserved microbial mats showing chain-like aggregates of pyrite grains, we observed a total of sixty-two morphotaxa including nine presumed prokaryotes, thirty-five possible prokaryotes or eukaryotes, fifteen unambiguous species of eukaryotes - ornamented and process-bearing acritarchs, multicellular morphotaxon, putative VSMs, large budding vesicles, and vesicles with a sophisticated excystment structure: the pylome - and three remains of structured kerogen. Here, we combined the geological context (sedimentological features and lithofacies), iron speciation (n = 156) - with the aim of reconstructing palaeoredox environmental conditions -, and microfossils quantitative analysis (n = 61). Sediments were deposited under shallow waters in pericratonic (western basin) and epicratonic (eastern basin

  13. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways.

  14. A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis.

    Science.gov (United States)

    Ramesh, Marilee A; Malik, Shehre-Banoo; Logsdon, John M

    2005-01-26

    Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.

  15. Hilar mossy cell circuitry controlling dentate granule cell excitability

    Directory of Open Access Journals (Sweden)

    Seiichiro eJinde

    2013-02-01

    Full Text Available Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability – the dormant basket cell and the irritable mossy cell hypotheses. The dormant basket cell hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The irritable mossy cell hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus.

  16. Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense.

    Directory of Open Access Journals (Sweden)

    Carsten Matz

    Full Text Available Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

  17. Remote Control of T Cell Activation Using Magnetic Janus Particles.

    Science.gov (United States)

    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  18. Cell density monitoring and control of microencapsulated CHO cell cultures

    OpenAIRE

    Cole, Harriet Emma

    2015-01-01

    Though mammalian cells play a key role in the manufacturing of recombinant glycosylated proteins, cell cultures and productivity are limited by the lack of suitable systems to enable stable perfusion culture. Microencapsulation, or entrapping cells within a semi-permeable membrane, offers the potential to generate high cell density cultures and improve the productivity by mimicking the cells natural environment. However, the cells being secluded by the microcapsules membrane are difficult to ...

  19. Simplified Load-Following Control for a Fuel Cell System

    Science.gov (United States)

    Vasquez, Arturo

    2010-01-01

    A simplified load-following control scheme has been proposed for a fuel cell power system. The scheme could be used to control devices that are important parts of a fuel cell system but are sometimes characterized as parasitic because they consume some of the power generated by the fuel cells.

  20. Self-assembled nanofiber coatings for controlling cell responses

    NARCIS (Netherlands)

    Barros, Raquel C.; Gelens, Edith; Bulten, Erna; Tuin, Annemarie; de Jong, Menno R; Kuijer, Roel; van Kooten, Theo G

    Nanofibers are thought to enhance cell adhesion, growth, and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2 D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts, and

  1. Epigenetic control of embryonic stem cell fate

    DEFF Research Database (Denmark)

    Christophersen, Nicolaj Strøyer; Helin, Kristian

    2010-01-01

    Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also...

  2. Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Bolstad, D; Bolstad, E; Wright, D; Anderson, A

    2009-01-01

    Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

  3. Statistical characteristics of eukaryotic intron database

    Institute of Scientific and Technical Information of China (English)

    HE Miao; LI Jidong; ZHANG Shanghong

    2006-01-01

    A database called eukaryotic intron database (EID) was developed based on the data from GenBank.Studies on the statistical characteristics of EID show that there were 103,848 genes,478,484 introns,and 582,332 exons,with an average of 4.61 introns and 5.61 exons per gene.Introns of 40-120 nt in length were abundant in the database.Results of the statistical analysis on the data from nine model species showed that in eukaryotes,higher species do not necessarily have more introns or exons in a gene than lower species.Furthermore,characteristics of EID,such as intron phase,distribution of different splice sites,and the relationship between genome size and intron proportion or intron density,have been studied.

  4. Faster growth of the major prokaryotic versus eukaryotic CO2 fixers in the oligotrophic ocean.

    Science.gov (United States)

    Zubkov, Mikhail V

    2014-04-29

    Because maintenance of non-scalable cellular components--membranes and chromosomes--requires an increasing fraction of energy as cell size decreases, miniaturization comes at a considerable energetic cost for a phytoplanktonic cell. Consequently, if eukaryotes can use their superior energetic resources to acquire nutrients with more or even similar efficiency compared with prokaryotes, larger unicellular eukaryotes should be able to achieve higher growth rates than smaller cyanobacteria. Here, to test this hypothesis, we directly compare the intrinsic growth rates of phototrophic prokaryotes and eukaryotes from the equatorial to temperate South Atlantic using an original flow cytometric (14)CO2-tracer approach. At the ocean basin scale, cyanobacteria double their biomass twice as frequently as the picoeukaryotes indicating that the prokaryotes are faster growing CO2 fixers, better adapted to phototrophic living in the oligotrophic open ocean-the most extensive biome on Earth.

  5. Lithium-Ion Cell Charge-Control Unit Developed

    Science.gov (United States)

    Reid, Concha M.; Manzo, Michelle A.; Buton, Robert M.; Gemeiner, Russel

    2005-01-01

    A lithium-ion (Li-ion) cell charge-control unit was developed as part of a Li-ion cell verification program. This unit manages the complex charging scheme that is required when Li-ion cells are charged in series. It enables researchers to test cells together as a pack, while allowing each cell to charge individually. This allows the inherent cell-to-cell variations to be addressed on a series string of cells and reduces test costs substantially in comparison to individual cell testing.

  6. Construction of eukaryotic expression vecter for human ski gene and observation of its role in promoting cell proliferation%人ski基因真核表达载体的构建及促细胞增殖作用

    Institute of Scientific and Technical Information of China (English)

    彭艳; 李平; 刘苹; 陈磊; 赵晓光; 周元国

    2011-01-01

    背景:c-Ski 既可促进组织修复,又能降低瘢痕形成,有望成为一个全新的基因治疗药物.目的:构建人ski 基因的真核表达载体,并对其促成纤维细胞增殖效果进行验证.方法:RT-PCR 法从人包皮培养原代成纤维细胞总RNA 中扩增出人ski 基因,连同真核表达启动子CMV 克隆到pUC118 载体上,酶切和测序验证后,应用脂质体将其转染到培养的大鼠皮肤成纤维细胞中.结果与结论:酶切和测序结果证实表达质粒构建成功,Western blot 显示Ski 蛋白在转染细胞中成功表达,且可显著提高转染细胞的增殖效应,说明以pUC118 为骨架的重组ski 表达质粒具有促进大鼠皮肤成纤维细胞增殖的作用.%BACKGROUND: c-Ski can promote tissue repair and alleviate scar formation, which is expected to be a new gene therapeutic drugs. OBJECTIVE: To construct eukaryotic expression plasmid of human ski gene and to investigate its proliferative role in fibroblasts. METHODS: The ski gene was amplified by polymerase chain reaction (PCR) from human foreskin cultured fibroblast and was sub-cloned into pUC118 with CMV eukaryotic promoter. The recombinant pUC118-Ski plasmids were transfected into rat skin fibroblasts using lipofectamine TM 2000 and then its protein expression and effect of the proliferation were detected. RESULTS AND CONCLUSION: pUC118-Ski vector was constructed successfully which was confirmed by restriction endonuclease digestion and DNA sequencing. It expressed successfully and significantly improved the effect of the proliferation in transfected fibroblasts. Recombinant eukaryotic expression plasmid pUC118-Ski can improve the proliferation of rat skin fibroblasts.

  7. Insights into the Initiation of Eukaryotic DNA Replication.

    Science.gov (United States)

    Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

    2015-01-01

    The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

  8. Insights into the Initiation of Eukaryotic DNA Replication

    Science.gov (United States)

    Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

    2015-01-01

    The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2–7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2–7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2–7 complex. Sld3 recruits Cdc45 to Mcm2–7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2–7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2–7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted. PMID:26710261

  9. An Evolutionary Network of Genes Present in the Eukaryote Common Ancestor Polls Genomes on Eukaryotic and Mitochondrial Origin

    OpenAIRE

    Thiergart, T.; Landan, G; Schenk, M.; Dagan, T.; Martin, W F

    2012-01-01

    To test the predictions of competing and mutually exclusive hypotheses for the origin of eukaryotes, we identified from a sample of 27 sequenced eukaryotic and 994 sequenced prokaryotic genomes 571 genes that were present in the eukaryote common ancestor and that have homologues among eubacterial and archaebacterial genomes. Maximum-likelihood trees identified the prokaryotic genomes that most frequently contained genes branching as the sister to the eukaryotic nuclear homologues. Among the a...

  10. Origin of eukaryotes from within archaea, archaeal eukaryome and bursts of gene gain: eukaryogenesis just made easier?

    Science.gov (United States)

    Koonin, Eugene V

    2015-09-26

    The origin of eukaryotes is a fundamental, forbidding evolutionary puzzle. Comparative genomic analysis clearly shows that the last eukaryotic common ancestor (LECA) possessed most of the signature complex features of modern eukaryotic cells, in particular the mitochondria, the endomembrane system including the nucleus, an advanced cytoskeleton and the ubiquitin network. Numerous duplications of ancestral genes, e.g. DNA polymerases, RNA polymerases and proteasome subunits, also can be traced back to the LECA. Thus, the LECA was not a primitive organism and its emergence must have resulted from extensive evolution towards cellular complexity. However, the scenario of eukaryogenesis, and in particular the relationship between endosymbiosis and the origin of eukaryotes, is far from being clear. Four recent developments provide new clues to the likely routes of eukaryogenesis. First, evolutionary reconstructions suggest complex ancestors for most of the major groups of archaea, with the subsequent evolution dominated by gene loss. Second, homologues of signature eukaryotic proteins, such as actin and tubulin that form the core of the cytoskeleton or the ubiquitin system, have been detected in diverse archaea. The discovery of this 'dispersed eukaryome' implies that the archaeal ancestor of eukaryotes was a complex cell that might have been capable of a primitive form of phagocytosis and thus conducive to endosymbiont capture. Third, phylogenomic analyses converge on the origin of most eukaryotic genes of archaeal descent from within the archaeal evolutionary tree, specifically, the TACK superphylum. Fourth, evidence has been presented that the origin of the major archaeal phyla involved massive acquisition of bacterial genes. Taken together, these findings make the symbiogenetic scenario for the origin of eukaryotes considerably more plausible and the origin of the organizational complexity of eukaryotic cells more readily explainable than they appeared until recently

  11. Illuminating traffic control for cell-division planes.

    Science.gov (United States)

    Robatzek, Silke

    2014-01-01

    When a plant cell divides, four related proteins control the trafficking of vesicles and ensure that cargo that is normally recycled to the plasma membrane is instead re-routed to the plane of cell division.

  12. MitoCOGs: clusters of orthologous genes from mitochondria and implications for the evolution of eukaryotes.

    Science.gov (United States)

    Kannan, Sivakumar; Rogozin, Igor B; Koonin, Eugene V

    2014-11-25

    Mitochondria are ubiquitous membranous organelles of eukaryotic cells that evolved from an alpha-proteobacterial endosymbiont and possess a small genome that encompasses from 3 to 106 genes. Accumulation of thousands of mitochondrial genomes from diverse groups of eukaryotes provides an opportunity for a comprehensive reconstruction of the evolution of the mitochondrial gene repertoire. Clusters of orthologous mitochondrial protein-coding genes (MitoCOGs) were constructed from all available mitochondrial genomes and complemented with nuclear orthologs of mitochondrial genes. With minimal exceptions, the mitochondrial gene complements of eukaryotes are subsets of the superset of 66 genes found in jakobids. Reconstruction of the evolution of mitochondrial genomes indicates that the mitochondrial gene set of the last common ancestor of the extant eukaryotes was slightly larger than that of jakobids. This superset of mitochondrial genes likely represents an intermediate stage following the loss and transfer to the nucleus of most of the endosymbiont genes early in eukaryote evolution. Subsequent evolution in different lineages involved largely parallel transfer of ancestral endosymbiont genes to the nuclear genome. The intron density in nuclear orthologs of mitochondrial genes typically is nearly the same as in the rest of the genes in the respective genomes. However, in land plants, the intron density in nuclear orthologs of mitochondrial genes is almost 1.5-fold lower than the genomic mean, suggestive of ongoing transfer of functional genes from mitochondria to the nucleus. The MitoCOGs are expected to become an important resource for the study of mitochondrial evolution. The nearly complete superset of mitochondrial genes in jakobids likely represents an intermediate stage in the evolution of eukaryotes after the initial, extensive loss and transfer of the endosymbiont genes. In addition, the bacterial multi-subunit RNA polymerase that is encoded in the jakobid

  13. Interactions of bacterial proteins with host eukaryotic ubiquitin pathways

    Directory of Open Access Journals (Sweden)

    Charlotte Averil Perrett

    2011-07-01

    Full Text Available Ubiquitination is a post-translational modification in which one or more 76 amino acid polypeptide ubiquitin molecules are covalently linked to the lysine residues of target proteins. Ubiquitination is the main pathway for protein degradation that governs a variety of eukaryotic cellular processes, including the cell cycle, vesicle trafficking, antigen presentation and signal transduction. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases including inflammatory and neurodegenerative disorders. Recent studies have revealed that viruses and bacterial pathogens exploit the host ubiquitination pathways to gain entry and to aid their survival/replication inside host cells. This review will summarize recent developments in understanding the biochemical and structural mechanisms utilized by bacterial pathogens to interact with the host ubiquitination pathways.

  14. The prokaryotic V4R domain is the likely ancestor of a key component of the eukaryotic vesicle transport system.

    Science.gov (United States)

    Podar, Mircea; Wall, Mark A; Makarova, Kira S; Koonin, Eugene V

    2008-01-25

    Intracellular vesicle traffic that enables delivery of proteins between the endoplasmic reticulum, Golgi and various endosomal subcompartments is one of the hallmarks of the eukaryotic cell. Its evolutionary history is not well understood but the process itself and the core vesicle traffic machinery are believed to be ancient. We show here that the 4-vinyl reductase (V4R) protein domain present in bacteria and archaea is homologous to the Bet3 subunit of the TRAPP1 vesicle-tethering complex that is conserved in all eukaryotes. This suggests, for the first time, a prokaryotic origin for one of the key eukaryotic trafficking proteins.

  15. miR-133a真核表达载体的构建及转染大鼠心肌H9C2细胞%Construction of miR-133a eukaryotic expression vector and transfection into H9C2 cell

    Institute of Scientific and Technical Information of China (English)

    张洪涛; 张赢予; 周艳芳; 王好; 郑枫; 张国辉

    2011-01-01

    Objective: To construct miR-133a eukaryotic expression vector and transfect it into H9C2 cell. Methods: Design and synthetize the PCR templa-primer. Ligate the pre-miR-133a with linearized PgenesIL-1. 1 by T4 DNA Ligase. The recombinants were identified by endonuclease digestion and sequenced. Transient transfect the perfect recombinants into H 9C2 cells by LipofectamineTM 2000 Reagent. Finally, detect transfection efficiency by flow cytometer and invert fluorescence microscope . Results: The miR-133a eukaryotic expression vectors were consistent with the design and the transfection efficiency of H9C2 cells was 70%. Conclusion: miR-133a eukaryotic expression vector was successfully constructed and transfected into H9C2 cell.%目的:构建miR-133a真核表达载体后,体外转染大鼠H9C2心肌细胞.方法:设计并合成DNA模板引物,用T4 DNA 连接酶将pre-miR-133a连接到线性化的PgenesIL-1.1质粒中,对重组质粒进行酶切及测序鉴定.以LipofectamineTM 2000 Reagent将鉴定正确的重组质粒瞬时转染H9C2细胞,用流式细胞仪及倒置荧光显微镜检测转染效率.结果:miR-133a真核表达载体符合设计要求,瞬时转染H9C2细胞的转染率达70%以上.结论:成功构建了miR-133a真核表达载体并转染至大鼠心肌H9C2细胞.

  16. Spatiotemporal control of cell-cell reversible interactions using molecular engineering

    Science.gov (United States)

    Shi, Peng; Ju, Enguo; Yan, Zhengqing; Gao, Nan; Wang, Jiasi; Hou, Jianwen; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

    2016-10-01

    Manipulation of cell-cell interactions has potential applications in basic research and cell-based therapy. Herein, using a combination of metabolic glycan labelling and bio-orthogonal click reaction, we engineer cell membranes with β-cyclodextrin and subsequently manipulate cell behaviours via photo-responsive host-guest recognition. With this methodology, we demonstrate reversible manipulation of cell assembly and disassembly. The method enables light-controllable reversible assembly of cell-cell adhesion, in contrast with previously reported irreversible effects, in which altered structure could not be reused. We also illustrate the utility of the method by designing a cell-based therapy. Peripheral blood mononuclear cells modified with aptamer are effectively redirected towards target cells, resulting in enhanced cell apoptosis. Our approach allows precise control of reversible cell-cell interactions and we expect that it will promote further developments of cell-based therapy.

  17. Control of transcription by cell size.

    Directory of Open Access Journals (Sweden)

    Chia-Yung Wu

    Full Text Available Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.

  18. An immunosurveillance mechanism controls cancer cell ploidy.

    Science.gov (United States)

    Senovilla, Laura; Vitale, Ilio; Martins, Isabelle; Tailler, Maximilien; Pailleret, Claire; Michaud, Mickaël; Galluzzi, Lorenzo; Adjemian, Sandy; Kepp, Oliver; Niso-Santano, Mireia; Shen, Shensi; Mariño, Guillermo; Criollo, Alfredo; Boilève, Alice; Job, Bastien; Ladoire, Sylvain; Ghiringhelli, François; Sistigu, Antonella; Yamazaki, Takahiro; Rello-Varona, Santiago; Locher, Clara; Poirier-Colame, Vichnou; Talbot, Monique; Valent, Alexander; Berardinelli, Francesco; Antoccia, Antonio; Ciccosanti, Fabiola; Fimia, Gian Maria; Piacentini, Mauro; Fueyo, Antonio; Messina, Nicole L; Li, Ming; Chan, Christopher J; Sigl, Verena; Pourcher, Guillaume; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Lazar, Vladimir; Penninger, Josef M; Madeo, Frank; López-Otín, Carlos; Smyth, Mark J; Zitvogel, Laurence; Castedo, Maria; Kroemer, Guido

    2012-09-28

    Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

  19. Eukaryotic Replisome Components Cooperate to Process Histones During Chromosome Replication

    Directory of Open Access Journals (Sweden)

    Magdalena Foltman

    2013-03-01

    Full Text Available DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.

  20. Eukaryotic replisome components cooperate to process histones during chromosome replication.

    Science.gov (United States)

    Foltman, Magdalena; Evrin, Cecile; De Piccoli, Giacomo; Jones, Richard C; Edmondson, Rick D; Katou, Yuki; Nakato, Ryuichiro; Shirahige, Katsuhiko; Labib, Karim

    2013-03-28

    DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.