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...

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...

Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution.

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Ajeet Mandal

Full Text Available The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro and/or PPG (Pro-Pro-Gly-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids, only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution.

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

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

Science.gov (United States)

Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2009-01-16

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

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

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Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

2012-01-01

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. PMID:22500084

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

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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.

System-level modeling and simulation of the cell culture microfluidic biochip ProCell

DEFF Research Database (Denmark)

Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

2010-01-01

Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

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

2016-01-01

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. PMID:26907343

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

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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.

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. © 2014 Elsevier Inc. All rights reserved.

Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

Science.gov (United States)

Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

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

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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.

Signaling mechanisms of apoptosis-like programmed cell death in unicellular eukaryotes.

Science.gov (United States)

Shemarova, Irina V

2010-04-01

In unicellular eukaryotes, apoptosis-like cell death occurs during development, aging and reproduction, and can be induced by environmental stresses and exposure to toxic agents. The essence of the apoptotic machinery in unicellular organisms is similar to that in mammals, but the apoptotic signal network is less complex and of more ancient origin. The review summarizes current data about key apoptotic proteins and mechanisms of the transduction of apoptotic signals by caspase-like proteases and mitochondrial apoptogenic proteins in unicellular eukaryotes. The roles of receptor-dependent and receptor-independent caspase cascades are reviewed. 2010 Elsevier Inc. All rights reserved.

Are maternal mitochondria the selfish entities that are masters of the cells of eukaryotic multicellular organisms?

Science.gov (United States)

Barlow, Peter W; Baldelli, E; Baluška, Frantisek

2009-01-01

The Energide concept, as well as the endosymbiotic theory of eukaryotic cell organization and evolution, proposes that present-day cells of eukaryotic organisms are mosaics of specialized and cooperating units, or organelles. Some of these units were originally free-living prokaryotes, which were engulfed during evolutionary time. Mitochondria represent one of these types of previously independent organisms, the Energide, is another type. This new perspective on the organization of the cell has been further expanded to reveal the concept of a public milieu, the cytosol, in which Energides and mitochondria live, each with their own private internal milieu. The present paper discusses how the endosymbiotic theory implicates a new hypothesis about the hierarchical and communicational organization of the integrated prokaryotic components of the eukaryotic cell and provides a new angle from which to consider the theory of evolution and its bearing upon cellular complexity. Thus, it is proposed that the “selfish gene” hypothesis of Dawkins1 is not the only possible perspective for comprehending genomic and cellular evolution. Our proposal is that maternal mitochondria are the selfish “master” entities of the eukaryotic cell with respect not only to their propagation from cell-to-cell and from generation-to-generation but also to their regulation of all other cellular functions. However, it should be recognized that the concept of “master” and “servant” cell components is a metaphor; in present-day living organisms their organellar components are considered to be interdependent and inseparable. PMID:19513277

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

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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.

Transfer of DNA from Bacteria to Eukaryotes

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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.

Do lipids shape the eukaryotic cell cycle?

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Furse, Samuel; Shearman, Gemma C

2018-01-01

Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

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Ines Teichert

2014-09-01

Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

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Andy Hesketh

2017-07-01

Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

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Baig, Abdul Mannan; Khan, Naveed Ahmed; Abbas, Farhat

2015-03-01

Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease.

Molecular and cellular characterization of the tomato pollen profilin, LePro1.

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Long-Xi Yu

Full Text Available Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton in all eukaryotic cells. We previously cloned a profilin gene, designated as LePro1 from tomato pollen. To understand its biological role, in the present study, we investigated the temporal and spatial expression of LePro1 during pollen development and found that the transcript was only detected at late stages during microsporogenesis and pollen maturation. Using antisense RNA, we successfully knocked down the expression of LePro1 in tomato plants using stable transformation, and obtained two antisense lines, A2 and A3 showing significant down-regulation of LePro1 in pollen resulting in poor pollen germination and abnormal pollen tube growth. A disorganized F-actin distribution was observed in the antisense pollen. Down-regulation of LePro1 also appeared to affect hydration of pollen deposited on the stigma and arrested pollen tube elongation in the style, thereby affecting fertilization. Our results suggest that LePro1 in conjunction with perhaps other cytoskeletal proteins, plays a regulatory role in the proper organization of F-actin in tomato pollen tubes through promoting actin assembly. Down-regulation of LePro1 leads to interruption of actin assembly and disorganization of the actin cytoskeleton thus arresting pollen tube growth. Based on the present and previous studies, it is likely that a single transcript of profilin gives rise to multiple forms displaying multifunctionality in tomato pollen.

Chitosan drives anti-inflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation

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MI Oliveira

2012-07-01

Full Text Available Macrophages and dendritic cells (DC share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch, with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-β1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1β levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tongfang Tang

Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

Science.gov (United States)

Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

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

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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

File list: ALL.Bld.20.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Bld.20.AllAg.Pro-B_cells mm9 All antigens Blood Pro-B cells SRX1553109,SRX15531...3,SRX1143907 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bld.20.AllAg.Pro-B_cells.bed ...

Proline oxidase silencing induces proline-dependent pro-survival pathways in MCF-7 cells

Science.gov (United States)

Zareba, Ilona; Celinska-Janowicz, Katarzyna; Surazynski, Arkadiusz; Miltyk, Wojciech; Palka, Jerzy

2018-01-01

Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process. PMID:29568391

File list: His.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available His.Bld.50.AllAg.Pro-B_cells mm9 Histone Blood Pro-B cells SRX668836,SRX1184113,SRX...09,SRX759800,SRX1143916,SRX1143902 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.50.AllAg.Pro-B_cells.bed ...

File list: His.Bld.20.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available His.Bld.20.AllAg.Pro-B_cells mm9 Histone Blood Pro-B cells SRX668836,SRX1184113,SRX...9,SRX1143910,SRX1143916,SRX1143902 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.20.AllAg.Pro-B_cells.bed ...

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

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

International Nuclear Information System (INIS)

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.

Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

International Nuclear Information System (INIS)

Zhdanov, V. P.

2010-01-01

Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

File list: NoD.Bld.10.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NoD.Bld.10.AllAg.Pro-B_cells mm9 No description Blood Pro-B cells SRX1553109,SRX155...3108,SRX1553107,SRX1553110 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Bld.10.AllAg.Pro-B_cells.bed ...

File list: NoD.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NoD.Bld.50.AllAg.Pro-B_cells mm9 No description Blood Pro-B cells SRX1553109,SRX155...3108,SRX1553107,SRX1553110 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Bld.50.AllAg.Pro-B_cells.bed ...

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

Science.gov (United States)

Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

2015-03-01

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes

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Silke Leimkühler

2017-01-01

Full Text Available Modifications of transfer RNA (tRNA have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm5s2U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron–sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT. Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes.

On the Archaeal Origins of Eukaryotes and the Challenges of Inferring Phenotype from Genotype.

Science.gov (United States)

Dey, Gautam; Thattai, Mukund; Baum, Buzz

2016-07-01

If eukaryotes arose through a merger between archaea and bacteria, what did the first true eukaryotic cell look like? A major step toward an answer came with the discovery of Lokiarchaeum, an archaeon whose genome encodes small GTPases related to those used by eukaryotes to regulate membrane traffic. Although 'Loki' cells have yet to be seen, their existence has prompted the suggestion that the archaeal ancestor of eukaryotes engulfed the future mitochondrion by phagocytosis. We propose instead that the archaeal ancestor was a relatively simple cell, and that eukaryotic cellular organization arose as the result of a gradual transfer of bacterial genes and membranes driven by an ever-closer symbiotic partnership between a bacterium and an archaeon. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Backup pathways of NHEJ in cells of higher eukaryotes: Cell cycle dependence

International Nuclear Information System (INIS)

Iliakis, George

2009-01-01

DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs, DNA ligase IV, XRCC4 and XLF/Cernunnos (D-NHEJ) as central components. Work carried out in our laboratory and elsewhere shows that when this pathway is chemically or genetically compromised, cells do not shunt DSBs to homologous recombination repair (HRR) but instead use another form of NHEJ operating as a backup (B-NHEJ). Here I review our efforts to characterize this repair pathway and discuss its dependence on the cell cycle as well as on the growth conditions. I present evidence that B-NHEJ utilizes ligase III, PARP-1 and histone H1. When B-NHEJ is examined throughout the cell cycle, significantly higher activity is observed in G2 phase that cannot be attributed to HRR. Furthermore, the activity of B-NHEJ is compromised when cells enter the plateau phase of growth. Together, these observations uncover a repair pathway with unexpected biochemical constitution and interesting cell cycle and growth factor regulation. They generate a framework for investigating the mechanistic basis of HRR contribution to DSB repair.

David and Goliath: chemical perturbation of eukaryotes by bacteria.

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Ho, Louis K; Nodwell, Justin R

2016-03-01

Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.

File list: InP.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available InP.Bld.50.AllAg.Pro-B_cells mm9 Input control Blood Pro-B cells SRX1143898,SRX6688...12,SRX759802,SRX759803,SRX1143899,SRX1143900,SRX1143913,SRX1143907 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Bld.50.AllAg.Pro-B_cells.bed ...

Endothelial cells provide a notch-dependent pro-tumoral niche for enhancing breast cancer survival, stemness and pro-metastatic properties.

Directory of Open Access Journals (Sweden)

Pegah Ghiabi

Full Text Available Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44HighCD24Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.

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.

Structural and biomechanical basis of mitochondrial movement in eukaryotic cells

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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

Recovery of soil unicellular eukaryotes: an efficiency and activity analysis on the single cell level.

Science.gov (United States)

Lentendu, Guillaume; Hübschmann, Thomas; Müller, Susann; Dunker, Susanne; Buscot, François; Wilhelm, Christian

2013-12-01

Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Symbiosis and the origin of eukaryotic motility

Science.gov (United States)

Margulis, L.; Hinkle, G.

1991-01-01

Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

Suppression of pro-inflammatory T-cell responses by human mesothelial cells.

Science.gov (United States)

Lin, Chan-Yu; Kift-Morgan, Ann; Moser, Bernhard; Topley, Nicholas; Eberl, Matthias

2013-07-01

Human γδ T cells reactive to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) contribute to acute inflammatory responses. We have previously shown that peritoneal dialysis (PD)-associated infections with HMB-PP producing bacteria are characterized by locally elevated γδ T-cell frequencies and poorer clinical outcome compared with HMB-PP negative infections, implying that γδ T cells may be of diagnostic, prognostic and therapeutic value in acute disease. The regulation by local tissue cells of these potentially detrimental γδ T-cell responses remains to be investigated. Freshly isolated γδ or αβ T cells were cultured with primary mesothelial cells derived from omental tissue, or with mesothelial cell-conditioned medium. Stimulation of cytokine production and proliferation by peripheral T cells in response to HMB-PP or CD3/CD28 beads was assessed by flow cytometry. Resting mesothelial cells were potent suppressors of pro-inflammatory γδ T cells as well as CD4+ and CD8+ αβ T cells. The suppression of γδ T-cell responses was mediated through soluble factors released by primary mesothelial cells and could be counteracted by SB-431542, a selective inhibitor of TGF-β and activin signalling. Recombinant TGF-β1 but not activin-A mimicked the mesothelial cell-mediated suppression of γδ T-cell responses to HMB-PP. The present findings indicate an important regulatory function of mesothelial cells in the peritoneal cavity by dampening pro-inflammatory T-cell responses, which may help preserve the tissue integrity of the peritoneal membrane in the steady state and possibly during the resolution of acute inflammation.

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

Science.gov (United States)

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

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.

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

Mechanisms and regulation of DNA replication initiation in eukaryotes.

Science.gov (United States)

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor α signalling

International Nuclear Information System (INIS)

Taylor, Catherine A.; Sun Zhong; Cliche, Dominic O.; Ming, Hong; Eshaque, Bithi; Jin Songmu; Hopkins, Marianne T.; Thai, Boun; Thompson, John E.

2007-01-01

Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death

ProSAAS-derived peptides are differentially processed and sorted in mouse brain and AtT-20 cells.

Directory of Open Access Journals (Sweden)

Jonathan H Wardman

Full Text Available ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.

Emerging paradigms and questions on pro-angiogenic bone marrow-derived myelomonocytic cells.

Science.gov (United States)

Laurent, Julien; Touvrey, Cédric; Botta, Francesca; Kuonen, François; Ruegg, Curzio

2011-01-01

Cancer-related inflammation has emerged in recent years as a major event contributing to tumor angiogenesis, tumor progression and metastasis formation. Bone marrow-derived and inflammatory cells promote tumor angiogenesis by providing endothelial progenitor cells that differentiate into mature endothelial cells, and by secreting pro-angiogenic factors and remodeling the extracellular matrix to stimulate angiogenesis though paracrine mechanisms. Several bone marrow-derived myelonomocytic cells, including monocytes and macrophages, have been identified and characterized by several laboratories in recent years. While the central role of these cells in promoting tumor angiogenesis, tumor progression and metastasis is nowadays well established, many questions remain open and new ones are emerging. These include the relationship between their phenotype and function, the mechanisms of pro-angiogenic programming, their contribution to resistance to anti-angiogenic treatments and to metastasis and their potential clinical use as biomarkers of angiogenesis and anti-angiogenic therapies. Here, we will review phenotypical and functional aspects of bone marrow-derived myelonomocytic cells and discuss some of the current outstanding questions.

T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

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Vince N Montes

Full Text Available Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week. The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

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

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2007-03-01

computer models of cell cycle regulation in a variety of organisms, including yeast cells, amphibian embryos, bacterial cells and human cells. These...and meiosis ), but they do not nullify the central role played by irreversible, alternating START and FINISH transitions in the cell cycle. 32...AFRL-IF-RS-TR-2007-69 Final Technical Report March 2007 EUKARYOTIC CELL CYCLE AS A TEST CASE FOR MODELING CELLULAR REGULATION IN A

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

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Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

2012-01-01

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

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

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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.

Arsenic and Antimony Transporters in Eukaryotes

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Ewa Maciaszczyk-Dziubinska

2012-03-01

Full Text Available 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.

Arsenic and Antimony Transporters in Eukaryotes

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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. PMID:22489166

Changes in proHB-EGF expression after functional activation of the immune system cells

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T. O. Chudina

2017-12-01

Full Text Available The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197 and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.

Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms.

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Sobierajska, Katarzyna; Fabczak, Hanna; Fabczak, Stanisław

2006-06-01

Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

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Zhuang, Yuan; Cheng, Ping; Liu, Xiao-fei; Peng, Liu-sheng; Li, Bo-sheng; Wang, Ting-ting; Chen, Na; Li, Wen-hua; Shi, Yun; Chen, Weisan; Pang, Ken C; Zeng, Ming; Mao, Xu-hu; Yang, Shi-ming; Guo, Hong; Guo, Gang; Liu, Tao; Zuo, Qian-fei; Yang, Hui-jie; Yang, Liu-yang; Mao, Fang-yuan; Lv, Yi-pin; Zou, Quan-ming

2015-09-01

Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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

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

2016-01-01

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. PMID:27892499

THE EXPRESSION OF Bcl-2 AND PRO-CASPASE 3 IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

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Andrej Cör

2002-12-01

Full Text Available Background. Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and accounts for 6% of cancers worldwide. A better understanding of its biology could lead to improved treatment options. Generally, the goal of cancer treatment is to abolish cell proliferation and to induce necrotic or aptoptotic cell death. Apoptosis has been recognized as a key mechanism of tumour cell elimination. Different apoptotic signals converge to induce caspase cascade activation. Caspase 3 is the central executioner caspase and is necessary for effective apoptotic cell death. Bcl-2 protein family regulates apoptosis. The Bcl-2 protein itself is a product of a proto-oncogene and has an antiapoptotic action.Methods. In our study, the expression of Bcl-2 and pro-caspase 3 by immunohistochemistry in 28 HNSCC graded into well, moderately and poorly differentiated cancers were investigated.Results. Our results of Bcl-2 expression confirm and extend previous reports in which Bcl-2 over-expression has been recognised as an important parameter in HNSCC biological behaviour. Three of 28 tumours (11% showed significant Bcl-2 expression. Two of them were poorly and one was moderately differentiated. Pro-caspase 3 immunoreactivity was confined mainly to the cytoplasm. Absent or low pro-caspase 3 immunoreactivity was found only in 1 of 6 well differentiated and in 1of 10 moderately differentiated tumours in contrast to 5 of 12 poorly differentiated tumours. In six of 12 poorly differentiated tumours procasapse 3 immunoreactivity was strongly positive. In two cases hyperplastic epithelium was strongly positive in contrast to adjacent HNSCC in the same slide which was completely negative for pro-caspase 3.Conclusions. Our results indicate downregulation of pro-caspase 3 expression, especially in poorly differentiated HNSCC. Further studies are needed to test whether this is related to HNSCC behaviour and predict treatment outcome.

Evaluation by mass fragmentography of metabolic pathways of endogenous and exogenous compounds in eukaryote cell cultures

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Padieu, P.; Maume, B.F.

1977-01-01

Carbon-14 labelled compounds in cell cultures are used to establish the interconnections between different metabolic pathways as well as the competitive action of effectors on these different pathways. Analysis was performed by the GC-MS combination. Identification was carried out by comparison with the mass spectra of d9-TMS, 35 Cl-TMS and 37 Cl-TMS derivatizations of the culture extracts. Examples are given of the metabolic study of hormonal steroids and of safrale, a carcinogenic compound, by differentiated eukaryotic cells in cultures from the rat

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

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Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

2010-01-01

Research highlights: → TGFβ induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. → TGFβ induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. → TGFβ enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. → Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGFβ. → ADAM17 may play a crucial role in this TGFβ-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF�

The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

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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.

DNA repair and its coupling to DNA replication in eukaryotic cells. [UV, x ray

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Cleaver, J.E.

1978-01-01

This review article with 184 references presents the view that mammalian cells have one major repair system, excision repair, with many branches (nucleotide excision repair, base excision repair, crosslink repair, etc.) and a multiplicity of enzymes. Any particular carcinogen makes a spectrum of damaged sites and each kind of damage may be repaired by one or more branches of excision repair. Excision repair is rarely complete, except at very low doses, and eukaryotic cells survive and replicate DNA despite the presence of unrepaired damage. An alteration in a specific biochemical pathway seen in damaged or mutant cells will not always be the primary consequence of damage or of the biochemical defect of the cells. Detailed kinetic data are required to understand comprehensively the various facets of excision repair and replication. Correlation between molecular events of repair and cytological and cellular changes such as chromosomal damage, mutagenesis, transformation, and carcinogenesis are also rudimentary.

Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?

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Likhitpanichkul, Morakot; Torre, Olivia M; Gruen, Jadry; Walter, Benjamin A; Hecht, Andrew C; Iatridis, James C

2016-05-03

During intervertebral disc (IVD) injury and degeneration, annulus fibrosus (AF) cells experience large mechanical strains in a pro-inflammatory milieu. We hypothesized that TNF-α, an initiator of IVD inflammation, modifies AF cell mechanobiology via cytoskeletal changes, and interacts with mechanical strain to enhance pro-inflammatory cytokine production. Human AF cells (N=5, Thompson grades 2-4) were stretched uniaxially on collagen-I coated chambers to 0%, 5% (physiological) or 15% (pathologic) strains at 0.5Hz for 24h under hypoxic conditions with or without TNF-α (10ng/mL). AF cells were treated with anti-TNF-α and anti-IL-6. ELISA assessed IL-1β, IL-6, and IL-8 production and immunocytochemistry measured F-actin, vinculin and α-tubulin in AF cells. TNF-α significantly increased AF cell pro-inflammatory cytokine production compared to basal conditions (IL-1β:2.0±1.4-84.0±77.3, IL-6:10.6±9.9-280.9±214.1, IL-8:23.9±26.0-5125.1±4170.8pg/ml for basal and TNF-α treatment, respectively) as expected, but mechanical strain did not. Pathologic strain in combination with TNF-α increased IL-1β, and IL-8 but not IL-6 production of AF cells. TNF-α treatment altered F-actin and α-tubulin in AF cells, suggestive of altered cytoskeletal stiffness. Anti-TNF-α (infliximab) significantly inhibited pro-inflammatory cytokine production while anti-IL-6 (atlizumab) did not. In conclusion, TNF-α altered AF cell mechanobiology with cytoskeletal remodeling that potentially sensitized AF cells to mechanical strain and increased TNF-α-induced pro-inflammatory cytokine production. Results suggest an interaction between TNF-α and mechanical strain and future mechanistic studies are required to validate these observations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Archaeal “Dark Matter” and the Origin of Eukaryotes

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Williams, Tom A.; Embley, T. Martin

2014-01-01

Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis. PMID:24532674

The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

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Pourgholaminejad, Arash; Aghdami, Nasser; Baharvand, Hossein; Moazzeni, Seyed Mohammad

2016-09-01

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders. Copyright © 2016. Published by Elsevier Ltd.

Origin of phagotrophic eukaryotes as social cheaters in microbial biofilms

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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

Identifying activated T cells in reconstituted RAG deficient mice using retrovirally transduced Pax5 deficient pro-B cells.

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Nadesan Gajendran

Full Text Available Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP and a far-red fluorescent protein from Heteractis crispa (HcRed. LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deficient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells.

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

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Dau, Andressa Minussi Pereira; da Silva, Eduardo Pradebon; da Rosa, Paulo Roberto Antunes; Bastiani, Felipe Tusi; Gutierrez, Karina; Ilha, Gustavo Freitas; Comim, Fabio Vasconcellos; Gonçalves, Paulo Bayard Dias

2016-07-01

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (Pmeiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (Pmeiosis in cattle. Copyright © 2016 Elsevier Inc. All rights reserved.

Mathematical model of reproductive death of irradiated eukaryotic cells, which considers saturation of DNA reparation system

International Nuclear Information System (INIS)

Knyigavko, V.G.; Ponomarenko, N.S.; Meshcheryakova, O.P.; Protasenya, S.Yu.

2009-01-01

A mathematical model of the processes determining reproductive death of the exposed cells was built. The model takes into account the phenomenon of saturation of the system of DNA radiation lesion reparation and structural functional peculiarities of chromatin structure in eukaryotes. The problem of assessment of the model parameters using experimental data was discussed.

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells.

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Driscoll, Timothy P; Verhoeve, Victoria I; Guillotte, Mark L; Lehman, Stephanie S; Rennoll, Sherri A; Beier-Sexton, Magda; Rahman, M Sayeedur; Azad, Abdu F; Gillespie, Joseph J

2017-09-26

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia ( Alphaproteobacteria ; Rickettsiaceae ). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. IMPORTANCE A hallmark of obligate intracellular

Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

Science.gov (United States)

Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

2015-02-10

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Energy Technology Data Exchange (ETDEWEB)

Ebi, Masahide [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan)

2010-11-19

Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

Pro-angiogenic cell-based therapy for the treatment of ischemic cardiovascular diseases.

Science.gov (United States)

Silvestre, Jean-Sébastien

2012-10-01

Pro-angiogenic cell therapy has emerged as a promising option to treat patients with acute myocardial infarction or with critical limb ischemia. Exciting pre-clinical studies have prompted the initiation of numerous clinical trials based on administration of stem/progenitor cells with pro-angiogenic potential. Most of the clinical studies performed so far have used bone marrow-derived or peripheral blood-derived mononuclear cells and showed, overall, a modest but significant benefit on tissue remodeling and function in patients with ischemic diseases. These mixed results pave the way for the development of strategies to overcome the limitation of autologous cell therapy and to propose more efficient approaches. Such strategies include pretreatment of cells with activators to augment cell recruitment and survival in the ischemic target area and/or the improvement of cell functions such as their paracrine ability to release proangiogenic factors and vasoactive molecules. In addition, efforts should be directed towards stimulation of both angiogenesis and vessel maturation, the development of a composite product consisting of stem/progenitor cells encapsulated in a biomaterial and the use of additional sources of regenerative cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Avian leukosis virus is a versatile eukaryotic platform for polypeptide display

International Nuclear Information System (INIS)

Khare, Pranay D.; Russell, Stephen J.; Federspiel, Mark J.

2003-01-01

Display technology refers to methods of generating libraries of modularly coded biomolecules and screening them for particular properties. Retroviruses are good candidates to be a eukaryotic viral platform for the display of polypeptides synthesized in eukaryotic cells. Here we demonstrate that avian leukosis virus (ALV) provides an ideal platform for display of nonviral polyaeptides expressed in a eukaryotic cell substrate. Different sizes of polypeptides were genetically fused to the extreme N-terminus of the ALV envelope glycoprotein in an ALV infectious clone containing an alkaline phosphatase reporter gene. The chimeric envelope glycoproteins were efficiently incorporated into virions and were stably displayed on the surface of the virions through multiple virus replication cycles. The foreign polypeptides did not interfere with the attachment and entry functions of the underlying ALV envelope glycoproteins. The displayed polypeptides were fully functional and could efficiently mediate attachment of the recombinant viruses to their respective cognate receptors. This study demonstrates that ALV is an ideal display platform for the generation and selection of libraries of polypeptides where there is a need for expression, folding, and posttranslational modification in the endoplasmic reticulum of eukaryotic cells

Activation of Pro-uPA is Critical for Initial Escape from the Primary Tumor and Hematogenous Dissemination of Human Carcinoma Cells

DEFF Research Database (Denmark)

Bekes, Erin; Deryugina, Elena; Kuprianova, Tatyana

2011-01-01

disseminating variant of the human PC-3 prostate carcinoma cell line, PC-hi/diss, as a prototype of aggressive carcinomas to investigate the mechanisms whereby pro-uPA activation and uPA-generated plasmin functionally contribute to specific stages of metastasis. The PC-hi/diss cells secrete and activate...... significant amounts of pro-uPA, leading to efficient generation of plasmin in solution and at the cell surface. In a mouse orthotopic xenograft model, treatment with the specific pro-uPA activation-blocking antibody mAb-112 significantly inhibited local invasion and distant metastasis of the PC-hi/diss cells....... To mechanistically examine the uPA/plasmin-mediated aspects of tumor cell dissemination, the anti pro-uPA mAb-112 and the potent serine protease inhibitor, aprotinin, were utilized in parallel in a number of in vivo assays modeling various rate-limiting steps in early metastatic spread. Our findings demonstrate...

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

Anionic lipids and the maintenance of membrane electrostatics in eukaryotes.

Science.gov (United States)

Platre, Matthieu Pierre; Jaillais, Yvon

2017-02-01

A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells.

Compositional patterns in the genomes of unicellular eukaryotes.

Science.gov (United States)

Costantini, Maria; Alvarez-Valin, Fernando; Costantini, Susan; Cammarano, Rosalia; Bernardi, Giorgio

2013-11-05

The genomes of multicellular eukaryotes are compartmentalized in mosaics of isochores, large and fairly homogeneous stretches of DNA that belong to a small number of families characterized by different average GC levels, by different gene concentration (that increase with GC), different chromatin structures, different replication timing in the cell cycle, and other different properties. A question raised by these basic results concerns how far back in evolution the compartmentalized organization of the eukaryotic genomes arose. In the present work we approached this problem by studying the compositional organization of the genomes from the unicellular eukaryotes for which full sequences are available, the sample used being representative. The average GC levels of the genomes from unicellular eukaryotes cover an extremely wide range (19%-60% GC) and the compositional patterns of individual genomes are extremely different but all genomes tested show a compositional compartmentalization. The average GC range of the genomes of unicellular eukaryotes is very broad (as broad as that of prokaryotes) and individual compositional patterns cover a very broad range from very narrow to very complex. Both features are not surprising for organisms that are very far from each other both in terms of phylogenetic distances and of environmental life conditions. Most importantly, all genomes tested, a representative sample of all supergroups of unicellular eukaryotes, are compositionally compartmentalized, a major difference with prokaryotes.

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

International Nuclear Information System (INIS)

Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

2014-01-01

Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

Energy Technology Data Exchange (ETDEWEB)

Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada); Klegeris, Andis [Department of Biology, University of British Columbia Okanagan, Kelowna, BC (Canada); Little, Jonathan P., E-mail: jonathan.little@ubc.ca [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada)

2014-03-28

Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

Directory of Open Access Journals (Sweden)

Mariela Rivera

Full Text Available Curcumin, an extract from the turmeric rhizome (Curcuma longa, is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12 and Poly (ADP-ribose polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

Science.gov (United States)

Rivera, Mariela; Ramos, Yanilda; Rodríguez-Valentín, Madeline; López-Acevedo, Sheila; Cubano, Luis A.; Zou, Jin; Zhang, Qiang; Wang, Guangdi

2017-01-01

Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses. PMID:28628644

Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties

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Lachlan M. Moldenhauer

2015-05-01

Full Text Available Circulating endothelial progenitor cells (EPCs provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3 strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs, namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo and thus may provide clinical utility for humans in the future.

Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Del Regno, Marisanta; Adesso, Simona; Popolo, Ada [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Quaroni, Andrea [Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University, Ithaca, NY 14853–6401 (United States); Autore, Giuseppina [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Severino, Lorella [Department of Pathology and Animal Health, Division of Toxicology, School of Veterinary Medicine, University of Naples “Federico II”, Via Delpino 1, 80137 Naples (Italy); Marzocco, Stefania, E-mail: smarzocco@unisa.it [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy)

2015-06-01

Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination.

An Evolutionary Framework for Understanding the Origin of Eukaryotes

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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.

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

Science.gov (United States)

Maschmeyer, Patrick; Petkau, Georg; Siracusa, Francesco; Zimmermann, Jakob; Zügel, Franziska; Kühl, Anja Andrea; Lehmann, Katrin; Schimmelpfennig, Sarah; Weber, Melanie; Haftmann, Claudia; Riedel, René; Bardua, Markus; Heinz, Gitta Anne; Tran, Cam Loan; Hoyer, Bimba Franziska; Hiepe, Falk; Herzog, Sebastian; Wittmann, Jürgen; Rajewsky, Nikolaus; Melchers, Fritz Georg; Chang, Hyun-Dong; Radbruch, Andreas; Mashreghi, Mir-Farzin

2018-05-01

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Anti- and pro-tumor functions of autophagy.

Science.gov (United States)

Morselli, Eugenia; Galluzzi, Lorenzo; Kepp, Oliver; Vicencio, José-Miguel; Criollo, Alfredo; Maiuri, Maria Chiara; Kroemer, Guido

2009-09-01

Autophagy constitutes one of the major responses to stress in eukaryotic cells, and is regulated by a complex network of signaling cascades. Not surprisingly, autophagy is implicated in multiple pathological processes, including infection by pathogens, inflammatory bowel disease, neurodegeneration and cancer. Both oncogenesis and tumor survival are influenced by perturbations of the molecular machinery that controls autophagy. Numerous oncoproteins, including phosphatidylinositol 3-kinase, Akt1 and anti-apoptotic members of the Bcl-2 family suppress autophagy. Conversely, several tumor suppressor proteins (e.g., Atg4c; beclin 1; Bif-1; BH3-only proteins; death-associated protein kinase 1; LKB1/STK11; PTEN; UVRAG) promote the autophagic pathway. This does not entirely apply to p53, one of the most important tumor suppressor proteins, which regulates autophagy in an ambiguous fashion, depending on its subcellular localization. Irrespective of the controversial role of p53, basal levels of autophagy appear to inhibit tumor development. On the contrary, chemotherapy- and metabolic stress-induced activation of the autophagic pathway reportedly contribute to the survival of formed tumors, thereby favoring resistance. In this context, autophagy inhibition would represent a major therapeutic target for chemosensitization. Here, we will review the current knowledge on the dual role of autophagy as an anti- and pro-tumor mechanism.

Pro-inflammatory cytokines derived from West Nile virus (WNV-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

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Nerurkar Vivek R

2010-10-01

Full Text Available Abstract Background WNV-associated encephalitis (WNVE is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death. Methods A transformed human neuroblastoma cell line, SK-N-SH, was infected with WNV at multiplicity of infection (MOI-1 and -5, and WNV replication kinetics and expression profile of key pro-inflammatory cytokines were analyzed by plaque assay, qRT-PCR, and ELISA. Cell death was measured in SK-N-SH cell line in the presence and absence of neutralizing antibodies against key pro-inflammatory cytokines using cell viability assay, TUNEL and flow cytometry. Further, naïve primary astrocytes were treated with UV-inactivated supernatant from mock- and WNV-infected SK-N-SH cell line and the activation of astrocytes was measured using flow cytometry and ELISA. Results WNV-infected SK-N-SH cells induced the expression of IL-1β, -6, -8, and TNF-α in a dose- and time-dependent manner, which coincided with increase in virus-induced cell death. Treatment of cells with anti-IL-1β or -TNF-α resulted in significant reduction of the neurotoxic effects of WNV. Furthermore treatment of naïve astrocytes with UV-inactivated supernatant from WNV-infected SK-N-SH cell line increased expression of glial fibrillary acidic protein and key inflammatory cytokines. Conclusion Our results for the first time suggest that neurons are one of the potential sources of pro-inflammatory cytokines in WNV-infected brain and these neuron-derived cytokines contribute to WNV

Colorimetric sensor for triphosphates and their application as a viable staining agent for prokaryotes and eukaryotes.

Science.gov (United States)

Ghosh, Amrita; Shrivastav, Anupama; Jose, D Amilan; Mishra, Sanjiv K; Chandrakanth, C K; Mishra, Sandhya; Das, Amitava

2008-07-15

The chromogenic complex 1 x Zn (where 1 is (E)-4-(4-dimethylamino-phenylazo)-N,N-bispyridin-2-ylmethyl-benzenesulfonamide) showed high affinity toward the phosphate ion in tetrabutylammonium phosphate in acetonitrile solution and could preferentially bind to adenosine triphosphate (ATP) in aqueous solution at physiological pH. This binding caused a visual change in color, whereas no such change was noticed with other related anions (adenosine monophosphate, adenosine diphosphate, pyrophosphate, and phosphate) of biological significance. Thus, 1 x Zn could be used as a staining agent for different biological cells through binding to the ATP, generated in situ by the mitochondria (in eukaryotes). For prokaryotes (bacteria) the cell membrane takes care of the cells' energy conversion, since they lack mitochondria. ATP is produced in their unique cell structure on the cell membrane, which is not found in any eukaryotes. These stained cells could be viewed with normal light microscopy. This reagent could even be used for distinguishing the gram-positive and the gram-negative bacteria (prokaryotes). This dye was found to be nonlipophilic in nature and nontoxic to living microbes (eukaryotes and prokaryotes). Further, stained cells were found to grow in their respective media, and this confirmed the maintenance of viability of the microbes even after staining, unlike with many other dyes available commercially.

Evolution of DNA replication protein complexes in eukaryotes and Archaea.

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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.

Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.

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Michael, S; Montag, M; Dott, W

2013-12-01

The objective of this study was to compare the toxicological effects of different source-related ambient PM10 samples in regard to their chemical composition. In this context we investigated airborne PM from different sites in Aachen, Germany. For the toxicological investigation human alveolar epithelial cells (A549) and murine macrophages (RAW264.7) were exposed from 0 to 96 h to increasing PM concentrations (0-100 μg/ml) followed by analyses of cell viability, pro-inflammatory and oxidative stress responses. The chemical analysis of these particles indicated the presence of 21 elements, water-soluble ions and PAHs. The toxicological investigations of the PM10 samples demonstrated a concentration- and time-dependent decrease in cell viability and an increase in pro-inflammatory and oxidative stress markers. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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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. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Arabinogalactan proteins have deep roots in eukaryotes

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Hervé, Cécile; Siméon, Amandine; Jam, Murielle

2016-01-01

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...

Silencing p75NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

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Ahmed Y Shanab

Full Text Available Accumulation of the nerve growth factor precursor (proNGF and its receptor p75NTR have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75NTR induce endothelial cell (EC death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75NTR were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75NTRshRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75NTR using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75NTR and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75NTR antagonist. Therefore, targeting p75NTR represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF.

Comparative genomics of phylogenetically diverse unicellular eukaryotes provide new insights into the genetic basis for the evolution of the programmed cell death machinery.

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Nedelcu, Aurora M

2009-03-01

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes--from ligands and receptors to executors of PCD--was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae--used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors.

Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana.

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Léonard, Simon; Chisholm, Joan; Laliberté, Jean-François; Sanfaçon, Hélène

2002-08-01

Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyvirus VPg and this interaction has been correlated with virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepovirus (Tomato ringspot virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m(7)GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the virus genome are discussed.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines

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Rabquer, Bradley J.; Ohara, Ray A.; Stinson, William A.; Campbell, Phillip L.; Amin, M. Asif; Balogh, Beatrix; Zakhem, George; Renauer, Paul A.; Lozier, Ann; Arasu, Eshwar; Haines, G. Kenneth; Kahaleh, Bashar; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.

2016-01-01

Objectives. Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. Methods. Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. Results. Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. Conclusion. Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc. PMID:26705326

Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

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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.

Eukaryotic ribosome display with in situ DNA recovery.

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He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Synthesis and tritium labeling of the highly potent mast cell-degranulating substance P analog H-Arg-Pro-Lys-Pro-NH-C sub 12 H sub 25

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Bienert, M.; Oehlke, J.; Niedrich, H. (Academy of Sciences of GDR, Berlin (Germany, F.R.). Inst. of Drug Research); Mittag, E. (Zentralinstitut fuer Kernforschung, Rossendorf (Germany, F.R.))

1990-12-01

Tritium labeling of the mast cell degranulating substance P analog H-Arg-Pro-Lys(3,4-{sup 3}H-Pro)-NH-C{sub 12}H{sub 25} by catalytic saturation of the dehydroproline (Dhp{sup 1}) double bond is described. Catalytic tritiation in water afforded the radioactive analog with a specific activity of 1.07 TBq/mmol. Tenfold enhancement of the catalyst-to-substrate ratio resulted in a reduced specific activity of 0.74 TBq/mmol. (author).

Blocking Modification of Eukaryotic Initiation 5A2 Antagonizes Cervical Carcinoma via Inhibition of RhoA/ROCK Signal Transduction Pathway.

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Liu, Xiaojun; Chen, Dong; Liu, Jiamei; Chu, Zhangtao; Liu, Dongli

2017-10-01

Cervical carcinoma is one of the leading causes of cancer-related death for female worldwide. Eukaryotic initiation factor 5A2 belongs to the eukaryotic initiation factor 5A family and is proposed to be a key factor involved in the development of diverse cancers. In the current study, a series of in vivo and in vitro investigations were performed to characterize the role of eukaryotic initiation factor 5A2 in oncogenesis and metastasis of cervical carcinoma. The expression status of eukaryotic initiation factor 5A2 in 15 cervical carcinoma patients was quantified. Then, the effect of eukaryotic initiation factor 5A2 knockdown on in vivo tumorigenicity ability, cell proliferation, cell cycle distribution, and cell mobility of HeLa cells was measured. To uncover the mechanism driving the function of eukaryotic initiation factor 5A2 in cervical carcinoma, expression of members within RhoA/ROCK pathway was detected, and the results were further verified with an RhoA overexpression modification. The level of eukaryotic initiation factor 5A2 in cervical carcinoma samples was significantly higher than that in paired paratumor tissues ( P cycle arrest ( P ROCK I, and ROCK II were downregulated. The above-mentioned changes in eukaryotic initiation factor 5A2 knockdown cells were alleviated by the overexpression of RhoA. The major findings outlined in the current study confirmed the potential of eukaryotic initiation factor 5A2 as a promising prognosis predictor and therapeutic target for cervical carcinoma treatment. Also, our data inferred that eukaryotic initiation factor 5A2 might function in carcinogenesis of cervical carcinoma through an RhoA/ROCK-dependent manner.

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.

The phylogenomic analysis of the anaphase promoting complex and its targets points to complex and modern-like control of the cell cycle in the last common ancestor of eukaryotes

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Brochier-Armanet Céline

2011-09-01

Full Text Available Abstract Background The Anaphase Promoting Complex or Cyclosome (APC/C is the largest member of the ubiquitin ligase [E3] family. It plays a crucial role in the control of the cell cycle and cell proliferation by mediating the proteolysis of key components by the proteasome. APC/C is made of a dozen subunits that assemble into a large complex of ~1.5 MDa, which interacts with various cofactors and targets. Results Using comparative genomic and phylogenetic approaches, we showed that 24 out of 37 known APC/C subunits, adaptors/co-activators and main targets, were already present in the Last Eukaryotic Common Ancestor (LECA and were well conserved to a few exceptions in all present-day eukaryotic lineages. The phylogenetic analysis of the 24 components inferred to be present in LECA showed that they contain a reliable phylogenetic signal to reconstruct the phylogeny of the domain Eucarya. Conclusions Taken together our analyses indicated that LECA had a complex and highly controlled modern-like cell cycle. Moreover, we showed that, despite what is generally assumed, proteins involved in housekeeping cellular functions may be a good complement to informational genes to study the phylogeny of eukaryotes.

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

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Vic Norris

2009-06-01

Full Text Available 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.

Cyr61/CCN1 and CTGF/CCN2 mediate the pro-angiogenic activity of VHL mutant renal carcinoma cells

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Chintalapudi, Mastan R.; Markiewicz, Margaret; Kose, Nurgun; Dammai, Vincent; Champion, Kristen J.; Hoda, Rana S.; Trojanowska, Maria; Hsu, Tien

2008-01-01

The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia inducible factor-alpha subunit (HIF-α). Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the pro-angiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor up-regulated in VHL mutant cells, the efficacy of anti-angiogenic therapy specifically targeting VEGF signaling remains modest. In this study we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared to VHL wild-type cells, VHL mutant RCC cells demonstrated a significantly increased pro-angiogenic activity, which correlated with increased secretion of Cyr61/CCN1, CTGF/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNAi knock-down experiments. Importantly, the pro-angiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2α function, the dominant HIF-α isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in renal cell carcinoma tissue samples showed that increased expression of these proteins correlates with loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple pro-angiogenic factors that function in parallel pathways. PMID:18212329

Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity

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Parfrey, Laura Wegener; Walters, William A.; Lauber, Christian L.; Clemente, Jose C.; Berg-Lyons, Donna; Teiling, Clotilde; Kodira, Chinnappa; Mohiuddin, Mohammed; Brunelle, Julie; Driscoll, Mark; Fierer, Noah; Gilbert, Jack A.; Knight, Rob

2014-06-19

Eukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across

Introduction of the human proα1(I) collagen gene into proα1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

International Nuclear Information System (INIS)

Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

1987-01-01

The Mov-13 mouse strain carries a retroviral insertion in the proα1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of proα2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse proα1(I) collagen gene into homozygous cell lines to assess whether the human or mouse proα1(I) chains can associate with the endogenous mouse proα2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human α1 chains and one mouse α2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both α1(I) and α2(I) chains in the human-mouse hybrid molecules were retarded, compared to the α(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse α1 and α2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human α chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected proα1(I) genes have on the synthesis, assembly, and function of collagen I

Structural-Functional Organization of the Eukaryotic Cell Nucleus and Transcription Regulation: Introduction to This Special Issue of Biochemistry (Moscow).

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Razin, S V

2018-04-01

This issue of Biochemistry (Moscow) is devoted to the cell nucleus and mechanisms of transcription regulation. Over the years, biochemical processes in the cell nucleus have been studied in isolation, outside the context of their spatial organization. Now it is clear that segregation of functional processes within a compartmentalized cell nucleus is very important for the implementation of basic genetic processes. The functional compartmentalization of the cell nucleus is closely related to the spatial organization of the genome, which in turn plays a key role in the operation of epigenetic mechanisms. In this issue of Biochemistry (Moscow), we present a selection of review articles covering the functional architecture of the eukaryotic cell nucleus, the mechanisms of genome folding, the role of stochastic processes in establishing 3D architecture of the genome, and the impact of genome spatial organization on transcription regulation.

Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

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Atteia, Ariane; van Lis, Robert; Tielens, Aloysius G M; Martin, William F

2013-02-01

Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems. Copyright © 2012 Elsevier B.V. All rights reserved.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

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Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Construction and identification of eukaryotic expression vector of pcDNA3-UHRF1

International Nuclear Information System (INIS)

Li Xinli; Zhu Ran; Zhu Wei; Fan Saijun; Meng Qinghui

2011-01-01

Objective: To generate eukaryotic expression vector of pcDNA3-UHRF1(ubiquitin-like, containing PHD and RING finger domains 1, UHRF1) and testify its expression in breast cancer cells MDA-MB-231. Methods: A 2.3 kb cDNA fragment was amplified from the total RNA of the human breast cancer cells MCF-7 by the RT-PCR method and was cloned into the plasmid pcDNA3. The vector was identified by the double digestion with restriction enzymes Kpn I and Xho I and was sequenced. The cDNA of UHRF1 was transfected into human breast cancer cells MDA-MB-231 by Lipofactamin2000. The positive clones were selected by G418. The expression of the UHRF1 was detected by RT-PCR and Western blot analysis. Results: The recombinant eukaryotic expression vector pcDNA3-UHRF1 was digested with Kpn I and BamH I, and the electrophoresis of the digested products showed two fragments; 2.3kb fragment of UHRF1 and 5.4 kb fragment of pcDNA3, and the sequence inserted was identical to the published sequence. The MDA-MB-231 cells transfected with the pcDNA3-UHRF1 plasmid expressed a high level of the UHRF1 mRNA and protein. Conclusion: The recombinant eukaryotic cell expression vector of pcDNA3-UHRF1 is constructed successfully. The recombinant plasmid pcDNA3-UHRF1 can provide a very useful tool and lay an important foundation for the research on the function of UHRF1. (authors)

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility*

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Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-01-01

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. “Knocking out” PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. PMID:26683373

Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

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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.

Defensins: antifungal lessons from eukaryotes

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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.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects

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Maria Eischen-Loges

2018-06-01

Full Text Available Background Electrical stimulation (ES has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES’s demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE. In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Methods Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. Results We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days after discontinuing ES exposure. Discussion This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This

Phenolic excipients of insulin formulations induce cell death, pro-inflammatory signaling and MCP-1 release

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Claudia Weber

2015-01-01

Insulin solutions displayed cytotoxic and pro-inflammatory potential caused by phenol or m-cresol. We speculate that during insulin pump therapy phenol and m-cresol might induce cell death and inflammatory reactions at the infusion site in vivo. Inflammation is perpetuated by release of MCP-1 by activated monocytic cells leading to enhanced recruitment of inflammatory cells. To minimize acute skin complications caused by phenol/m-cresol accumulation, a frequent change of infusion sets and rotation of the infusion site is recommended.

Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

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Harshini Chakravarthy

Full Text Available Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs. We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

A study of eukaryotic response mechanisms to atmospheric pressure cold plasma by using Saccharomyces cerevisiae single gene mutants

International Nuclear Information System (INIS)

Feng Hongqing; Wang Ruixue; Sun Peng; Wu Haiyan; Liu Qi; Li Fangting; Fang Jing; Zhang Jue; Zhu Weidong

2010-01-01

The mechanisms of eukaryotic cell response to cold plasma are studied. A series of single gene mutants of eukaryotic model organism Saccharomyces cerevisiae are used to compare their sensitivity to plasma treatment with the wild type. We examined 12 mutants in the oxidative stress pathway and the cell cycle pathway, in which 8 are found to be hypersensitive to plasma processing. The mutated genes' roles in the two pathways are analyzed to understand the biological response mechanisms of plasma treatment. The results demonstrate that genes from both pathways are needed for the eukaryotic cells to survive the complex plasma treatment.

The milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro attenuate arterial dysfunction in L-NAME-treated rats.

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Nonaka, Atsuko; Nakamura, Teppei; Hirota, Tatsuhiko; Matsushita, Akiko; Asakura, Masanori; Ohki, Kohji; Kitakaze, Masafumi

2014-08-01

Both endothelial dysfunction and arterial stiffness are surrogate markers of atherosclerosis and thus cardiovascular (CV) events. The milk-derived peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) inhibit angiotensin-converting enzyme, dilate blood vessels ex vivo and stimulate nitric oxide (NO) production in cells. In this study, we investigated the effects of either VPP or IPP on arterial function and on target organ damage in vivo. Male Wistar rats were treated with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 g l(-1)), L-NAME+VPP (0.3 g l(-1)) or L-NAME+IPP (0.3 g l(-1)) in their drinking water for 8 weeks. Plasma nitrite and nitrate (NOx) levels were significantly increased in normal Wistar rats after supplementation with either VPP or IPP but not in rats that were chronically treated with L-NAME. Acetylcholine-induced vasorelaxation in the thoracic aorta ring was impaired by L-NAME, whereas vasorelaxation was significantly greater in mice treated with L-NAME+VPP for 1 or 4 weeks or L-NAME+IPP for 4 weeks than in mice treated with L-NAME alone. Four weeks of treatment with either VPP or IPP attenuated the increase in pulse wave velocity (PWV) that was induced by L-NAME. Cardiac and renal damage were observed after 8 weeks of treatment with L-NAME, and either VPP or IPP attenuated this damage. These results show that VPP or IPP attenuates arterial dysfunction and suggest that milk-derived peptides might prevent CV damage.

Distinct gene number-genome size relationships for eukaryotes and non-eukaryotes: gene content estimation for dinoflagellate genomes.

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Yubo Hou

Full Text Available The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log(10-transformed protein-coding gene number (Y' versus log(10-transformed genome size (X', genome size in kbp were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Y' = ln(-46.200+22.678X', whereas non-eukaryotes a linear model, Y' = 0.045+0.977X', both with high significance (p0.91. Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%-1% compared to higher and relatively stable percentages in prokaryotes and viruses (97%-47%. The eukaryotic regression models project that the smallest dinoflagellate genome (3x10(6 kbp contains 38,188 protein-coding (40,086 total genes and the largest (245x10(6 kbp 87,688 protein-coding (92,013 total genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species.

Morphological and ecological complexity in early eukaryotic ecosystems.

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Javaux, E J; Knoll, A H; Walter, M R

2001-07-05

Molecular phylogeny and biogeochemistry indicate that eukaryotes differentiated early in Earth history. Sequence comparisons of small-subunit ribosomal RNA genes suggest a deep evolutionary divergence of Eukarya and Archaea; C27-C29 steranes (derived from sterols synthesized by eukaryotes) and strong depletion of 13C (a biogeochemical signature of methanogenic Archaea) in 2,700 Myr old kerogens independently place a minimum age on this split. Steranes, large spheroidal microfossils, and rare macrofossils of possible eukaryotic origin occur in Palaeoproterozoic rocks. Until now, however, evidence for morphological and taxonomic diversification within the domain has generally been restricted to very late Mesoproterozoic and Neoproterozoic successions. Here we show that the cytoskeletal and ecological prerequisites for eukaryotic diversification were already established in eukaryotic microorganisms fossilized nearly 1,500 Myr ago in shales of the early Mesoproterozoic Roper Group in northern Australia.

RNA Export through the NPC in Eukaryotes.

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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.

Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

International Nuclear Information System (INIS)

Verhaeghe, Catherine; Tabruyn, Sebastien P.; Oury, Cecile; Bours, Vincent; Griffioen, Arjan W.

2007-01-01

Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

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Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-02-19

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Heat stress-induced loss of eukaryotic initiation factor 5A (eIF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis.

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Takeuchi, Kana; Nakamura, Kazuyuki; Fujimoto, Masanori; Kaino, Seiji; Kondoh, Satoshi; Okita, Kiwamu

2002-02-01

Alterations of intracellular proteins during the process of heat stress-induced cell death of a human pancreatic cancer cell line, MIA PaCa-2, were investigated using two-dimensional gel electrophoresis (2-DE), agarose gel electrophoresis, and cell biology techniques. Incubation of MIA PaCa-2 at 45 degrees C for 30 min decreased the cell growth rate and cell viability without causing chromosomal DNA fragmentation. Incubation at 51 degrees C for 30 min suppressed cell growth and again led to death without DNA fragmentation. The cell death was associated with the loss of an intracellular protein of M(r) 17,500 and pI 5.2 on 2-DE gel. This protein was determined to be eukaryotic initiation factor SA (eIF-5A) by microsequencing of the N-terminal region of peptide fragments obtained by cyanogen bromide treatment of the protein blotted onto a polyvinylidene difluoride (PVDF) membrane. The sequences detected were QXSALRKNGFVVLKGRP and STSKTGXHGHAKVHLVGID, which were homologous with the sequence of eIF-5A from Gln 20 to Pro 36 and from Ser 43 to Asp 61, respectively. Furthermore, the result of sequencing suggested that the protein was an active form of hypusinated eIF-5A, because Lys 46 could be detected but not Lys 49, which is the site for hypusination. These results suggest that loss of the active form of eIF-5A is an important factor in the irreversible process of heat stress-induced death of MIA PaCa-2 cells.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

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Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

International Nuclear Information System (INIS)

Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu

2005-01-01

We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis

The mRNA expression of pro- and anti-inflammatory cytokines in T regulatory cells in children with type 1 diabetes.

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Maria Górska

2010-06-01

Full Text Available Type 1 diabetes mellitus (T1DM is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T regulatory cells (Tregs represent an active mechanism of suppressing autoreactive T cells that escape central tolerance. The aim of our study was to test the hypothesis that T regulatory cells express pro- and anti-inflammatory cytokines, elements of cytotoxicity and OX40/4-1BB molecules. The examined group consisted of 50 children with T1DM. Fifty two healthy individuals (control group were enrolled into the study. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD134 and anti-CD137. Concurrently with the flow cytometric assessment of Tregs we separated CD4+CD25+CD127dim/- cells for further mRNA analysis. mRNA levels for transcription factor FoxP3, pro- and anti-inflammatory cytokines (interferon gamma, interleukin-2, interleukin-4, interleukin-10, transforming growth factor beta1 and tumor necrosis factor alpha, activatory molecules (OX40, 4-1BB and elements of cytotoxicity (granzyme B, perforin 1 were determined by real-time PCR technique. We found no alterations in the frequency of CD4+CD25highCD127low cells between diabetic and control children. Treg cells expressed mRNA for pro- and anti-inflammatory cytokines. Lower OX40 and higher 4-1BB mRNA but not protein levels in Treg cells in diabetic patients compared to the healthy children were noted. Our observations confirm the presence of mRNA for pro- and anti-inflammatory cytokines in CD4+CD25+CD127dim/- cells in the peripheral blood of children with T1DM. Further studies with the goal of developing new strategies to potentiate Treg function in autoimmune diseases are warranted.

Construction and expression of eukaryotic expression vectors of full-length, amino-terminus and carboxyl-terminus Raf gene

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Zhuomin WANG

2008-06-01

Full Text Available Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were successfully constructed and can be expressed in 293T cells.

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

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Dominique Colinet

2007-12-01

Full Text Available Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.

An algorithm for detecting eukaryotic sequences in metagenomic ...

Indian Academy of Sciences (India)

species but also from accidental contamination from the genome of eukaryotic host cells. The latter scenario generally occurs in the case of host-associated metagenomes, e.g. microbes living in human gut. In such cases, one needs to identify and remove contaminating host DNA sequences, since the latter sequences will ...

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

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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.

[Merkel cell carcinoma: cutaneous manifestation of a highly malignant pre-/pro-B cell neoplasia? : Novel concept about the cellular origin of Merkel cell carcinoma].

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Sauer, C M; Chteinberg, E; Rennspiess, D; Kurz, A K; Zur Hausen, A

2017-03-01

Merkel cell carcinoma (MCC) is a relatively rare but highly malignant non-melanoma skin cancer of the elderly and immunosuppressed patients. The discovery of the Merkel cell polyomavirus (MCPyV) in 2008 significantly impacted the understanding of the etiopathogenesis of MCC. MCPyV is clonally integrated into the MCC genome and approximately 80% of MCC are MCPyV-positive. Recent results of clinical trials using blockade of the PD-1 immune modulatory pathway are promising for the future treatment of MCC. Despite this major progress of the past few years, the cellular origin of MCC still remains obscure. Based on histomorphology, gene expression profiling, and molecular analyses, we have recently hypothesized that MCC originates from pre‑/pro-B cells. Here we review putative cells of MCC, including Merkel cells, (epi‑)dermal stem cells, and pro‑/pre-B cells. In the present work, the focus is on the concept of pre‑/pro-B cells as the cellular origin of MCC, which might also impact the understanding of other human small cell malignancies of unknown cellular origin, such as small cell carcinomas of the lung and other anatomical locations. In addition, this concept might pave the way for novel treatment options, especially for advanced MCC.

Enzymes from Higher Eukaryotes for Industrial Biocatalysis

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Zhibin Liu

2004-01-01

Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

Pro-inflammatory stimulation of meniscus cells increases production of matrix metalloproteinases and additional catabolic factors involved in osteoarthritis pathogenesis

Science.gov (United States)

Stone, Austin V.; Loeser, Richard F.; Vanderman, Kadie S.; Long, David L.; Clark, Stephanie C.; Ferguson, Cristin M.

2014-01-01

Objective Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development. Design Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1β, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-κB was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-κB inhibitor, hypoestoxide. Results Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor gene expression was reduced. Expression of cytokines (IL-1α, IL-1β, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-κB and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production. Conclusions Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes. PMID:24315792

The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.).

Science.gov (United States)

Jefferson, Matthew; Whelband, Matthew; Mohorianu, Irina; Powell, Penny P

2014-01-01

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway.

NT-pro brain natriuretic peptide levels and the risk of death in the cooperative study of sickle cell disease.

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Machado, Roberto F; Hildesheim, Mariana; Mendelsohn, Laurel; Remaley, Alan T; Kato, Gregory J; Gladwin, Mark T

2011-08-01

Epidemiological studies support a hypothesis that pulmonary hypertension (PH) is a common complication of sickle cell disease (SCD) that is associated with a high risk of death and evolves as a complication of haemolytic anaemia. This fundamental hypothesis has been recently challenged and remains controversial. In order to further test this hypothesis in a large and independent cohort of SCD patients we obtained plasma samples from the Cooperative Study of Sickle Cell Disease (CSSCD) for analysis of a biomarker, N-terminal-pro brain natriuretic peptide (NT-proBNP), which is elevated in the setting of pulmonary arterial and venous hypertension. A NT-pro-BNP value previously identified to predict PH in adults with SCD was used to determine the association between the risk of mortality in 758 CSSCD participants (428 children and 330 adults). An abnormally high NT-proBNP level ≥160ng/l was present in 27·6% of adult SCD patients. High levels were associated with markers of haemolytic anaemia, such as low haemoglobin level (P<0·001), high lactate dehydrogenase (P<0·001), and high total bilirubin levels (P<0·007). A NT-proBNP level ≥160ng/l was an independent predictor of mortality (RR 6·24, 95% CI 2·9-13·3, P<0·0001). These findings provide further support for an association between haemolytic anaemia and cardiovascular complications in this patient population. © 2011 Blackwell Publishing Ltd.

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development.

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Shima, Naoko; Pederson, Kayla D

2017-08-01

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

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...

Regulated eukaryotic DNA replication origin firing with purified proteins.

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Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

International Nuclear Information System (INIS)

Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

2015-01-01

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

Origins and evolution of viruses of eukaryotes: The ultimate modularity

Energy Technology Data Exchange (ETDEWEB)

Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov [National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 (United States); Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu [Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (United States); Krupovic, Mart, E-mail: krupovic@pasteur.fr [Institut Pasteur, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, Paris 75015 (France)

2015-05-15

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

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

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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.

The pestivirus N terminal protease N(pro redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro..

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Matthew Jefferson

Full Text Available The N-terminal protease of pestiviruses, N(pro is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro, but not by mutant protein N(pro C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro and infection with Bovine Viral Diarrhea Virus (BVDV prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro and we show that, in common with many other viral proteins, N(pro targets mitochondria to inhibit apoptosis in response to cell stress. N(pro itself not only relocated to mitochondria but in addition, both N(pro and IRF3 associated with peroxisomes, with over 85% of N(pro puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro, and highlight the role of these organelles in the anti-viral pathway.

Conservation and Variability of Meiosis Across the Eukaryotes.

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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.

A novel pleiotropic effect of aspirin: Beneficial regulation of pro- and anti-inflammatory mechanisms in microglial cells.

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Kata, Diana; Földesi, Imre; Feher, Liliana Z; Hackler, Laszlo; Puskas, Laszlo G; Gulya, Karoly

2017-06-01

Aspirin, one of the most widely used non-steroidal anti-inflammatory drugs, has extensively studied effects on the cardiovascular system. To reveal further pleiotropic, beneficial effects of aspirin on a number of pro- and anti-inflammatory microglial mechanisms, we performed morphometric and functional studies relating to phagocytosis, pro- and anti-inflammatory cytokine production (IL-1β, tumor necrosis factor-α (TNF-α) and IL-10, respectively) and analyzed the expression of a number of inflammation-related genes, including those related to the above functions, in pure microglial cells. We examined the effects of aspirin (0.1mM and 1mM) in unchallenged (control) and bacterial lipopolysaccharide (LPS)-challenged secondary microglial cultures. Aspirin affected microglial morphology and functions in a dose-dependent manner as it inhibited LPS-elicited microglial activation by promoting ramification and the inhibition of phagocytosis in both concentrations. Remarkably, aspirin strongly reduced the pro-inflammatory IL-1β and TNF-α production, while it increased the anti-inflammatory IL-10 level in LPS-challenged cells. Moreover, aspirin differentially regulated the expression of a number of inflammation-related genes as it downregulated such pro-inflammatory genes as Nos2, Kng1, IL1β, Ptgs2 or Ccr1, while it upregulated some anti-inflammatory genes such as IL10, Csf2, Cxcl1, Ccl5 or Tgfb1. Thus, the use of aspirin could be beneficial for the prophylaxis of certain neurodegenerative disorders as it effectively ameliorates inflammation in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

[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.

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single molecule real-time sequencing.

Science.gov (United States)

Zhu, Shijia; Beaulaurier, John; Deikus, Gintaras; Wu, Tao; Strahl, Maya; Hao, Ziyang; Luo, Guanzheng; Gregory, James A; Chess, Andrew; He, Chuan; Xiao, Andrew; Sebra, Robert; Schadt, Eric E; Fang, Gang

2018-05-15

N6-methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes, however, methods for high resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes, and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single nucleotide and single molecule resolution. For human lymphoblastoid cells (hLCLs), joint analyses of SMRT sequencing and independent sequencing data suggest that putative m6dA events are enriched in the promoters of young, full length LINE-1 elements (L1s). These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes. Published by Cold Spring Harbor Laboratory Press.

Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands

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Leo Veenman

2016-06-01

Full Text Available The 18 kDa translocator protein (TSPO is highly 0conserved in eukaryotes and prokaryotes. Since its discovery in 1977, numerous studies established the TSPO’s importance for life essential functions. For these studies, synthetic TSPO ligands typically are applied. Tetrapyrroles present endogenous ligands for the TSPO. Tetrapyrroles are also evolutionarily conserved and regulate multiple functions. TSPO and tetrapyrroles regulate each other. In animals TSPO-tetrapyrrole interactions range from effects on embryonic development to metabolism, programmed cell death, response to stress, injury and disease, and even to life span extension. In animals TSPOs are primarily located in mitochondria. In plants TSPOs are also present in plastids, the nuclear fraction, the endoplasmic reticulum, and Golgi stacks. This may contribute to translocation of tetrapyrrole intermediates across organelles’ membranes. As in animals, plant TSPO binds heme and protoporphyrin IX. TSPO-tetrapyrrole interactions in plants appear to relate to development as well as stress conditions, including salt tolerance, abscisic acid-induced stress, reactive oxygen species homeostasis, and finally cell death regulation. In bacteria, TSPO is important for switching from aerobic to anaerobic metabolism, including the regulation of photosynthesis. As in mitochondria, in bacteria TSPO is located in the outer membrane. TSPO-tetrapyrrole interactions may be part of the establishment of the bacterial-eukaryote relationships, i.e., mitochondrial-eukaryote and plastid-plant endosymbiotic relationships.

Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

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Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

2015-03-05

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

Science.gov (United States)

Bolla, P A; Abraham, A G; Pérez, P F; de Los Angeles Serradell, M

2016-02-01

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

MitProNet: A knowledgebase and analysis platform of proteome, interactome and diseases for mammalian mitochondria.

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Jiabin Wang

Full Text Available Mitochondrion plays a central role in diverse biological processes in most eukaryotes, and its dysfunctions are critically involved in a large number of diseases and the aging process. A systematic identification of mitochondrial proteomes and characterization of functional linkages among mitochondrial proteins are fundamental in understanding the mechanisms underlying biological functions and human diseases associated with mitochondria. Here we present a database MitProNet which provides a comprehensive knowledgebase for mitochondrial proteome, interactome and human diseases. First an inventory of mammalian mitochondrial proteins was compiled by widely collecting proteomic datasets, and the proteins were classified by machine learning to achieve a high-confidence list of mitochondrial proteins. The current version of MitProNet covers 1124 high-confidence proteins, and the remainders were further classified as middle- or low-confidence. An organelle-specific network of functional linkages among mitochondrial proteins was then generated by integrating genomic features encoded by a wide range of datasets including genomic context, gene expression profiles, protein-protein interactions, functional similarity and metabolic pathways. The functional-linkage network should be a valuable resource for the study of biological functions of mitochondrial proteins and human mitochondrial diseases. Furthermore, we utilized the network to predict candidate genes for mitochondrial diseases using prioritization algorithms. All proteins, functional linkages and disease candidate genes in MitProNet were annotated according to the information collected from their original sources including GO, GEO, OMIM, KEGG, MIPS, HPRD and so on. MitProNet features a user-friendly graphic visualization interface to present functional analysis of linkage networks. As an up-to-date database and analysis platform, MitProNet should be particularly helpful in comprehensive studies of

Decreased Regulatory T Cells in Vulnerable Atherosclerotic Lesions: Imbalance between Pro- and Anti-Inflammatory Cells in Atherosclerosis

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Ilonka Rohm

2015-01-01

Full Text Available Atherosclerosis is a chronic inflammatory disease of the arterial wall in which presentation of autoantigens by dendritic cells (DCs leads to the activation of T cells. Anti-inflammatory cells like Tregs counterbalance inflammation in atherogenesis. In our study, human carotid plaque specimens were classified as stable (14 and unstable (15 according to established morphological criteria. Vessel specimens (n=12 without any signs of atherosclerosis were used as controls. Immunohistochemical staining was performed to detect different types of DCs (S100, fascin, CD83, CD209, CD304, and CD123, proinflammatory T cells (CD3, CD4, CD8, and CD161, and anti-inflammatory Tregs (FoxP3. The following results were observed: in unstable lesions, significantly higher numbers of proinflammatory cells like DCs, T helper cells, cytotoxic T cells, and natural killer cells were detected compared to stable plaques. Additionally, there was a significantly higher expression of HLA-DR and more T cell activation (CD25, CD69 in unstable lesions. On the contrary, unstable lesions contained significantly lower numbers of Tregs. Furthermore, a significant inverse correlation between myeloid DCs and Tregs was shown. These data suggest an increased inflammatory state in vulnerable plaques resulting from an imbalance of the frequency of local pro- and anti-inflammatory immune cells.

Accumulation of pro-cancer cytokines in the plasma fraction of stored packed red cells.

Science.gov (United States)

Benson, Douglas D; Beck, Adam W; Burdine, Marie S; Brekken, Rolf; Silliman, Christopher C; Barnett, Carlton C

2012-03-01

Perioperative blood transfusion has been linked to decreased survival in pancreatic cancer; however, the exact causal mechanism has not been elucidated. Allogeneic transfusions are known to expose patients to foreign cells and lipid mediators. We hypothesize that stored packed red cells (pRBCs) contain pro-cancer cytokines that augment tumor progression. We analyzed the plasma fraction of stored pRBCs for pro-cancer cytokines and evaluated the affect of both storage time and leukocyte reduction on these mediators. Chemiarray™ analysis for pro-cancer cytokines was performed on the acellular plasma fraction of stored leukocyte-reduced (LR) and non-leukocyte-reduced (NLR) pRBCs at day 1 (D.1-fresh) and day 42 (D.42-outdate) of storage. Elevated expression of monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), angiogenin, tumor necrosis factor-alpha (TNF-α), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) was found. Specific enzyme-linked immunosorbent assay was performed for each of these factors in LR and NLR blood at D.1, day 28, and D.42. Data were analyzed by ANOVA. A p value ≤ 0.05 was considered significant; N ≥ 4 per group. Migration assays were performed using inhibitors of EGF (gefitinib) and PDGF (imatinib) on murine pancreatic adenocarcinoma cells (Pan02) exposed to D.1 and D.42 LR and NLR plasma. Proliferation assays were performed on Pan02 cells to test the inhibition of PDGF. MCP-1 levels increased with storage time in LR blood, 86.3 ± 6.3 pg/ml at D.1 vs. 121.2 ± 6.1 pg/ml at D.42 (p = 0.007), and NLR blood, 78.2 ± 7.3 pg/ml at D.1 vs. 647.8 ± 220.7 pg/ml at D.42 (p = 0.02). RANTES levels are lower in LR compared to NLR stored blood, 3.0 ± 1.9 vs. 15.8 ± 0.7 pg/ml at D.42 (p Pro-cancer cytokines that can augment tumor progression were identified in pRBCs. Some of these factors are present in fresh blood. The soluble factors identified herein may represent

Tumour-Derived Interleukin-1 Beta Induces Pro-inflammatory Response in Human Mesenchymal Stem Cells

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Alajez, Nehad M; Al-toub, Mashael; Almusa, Abdulaziz

’ secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. Background Over the past several years, significant amount of research has emerged......, the goal of this study was to assess the cellular and molecular changes in MSCs in response to secreted factors present in conditioned media (CM) from a panel of human tumor cell lines covering a spectrum of human cancers (Breast, Prostate, Lung, colon, and head and neck). Research Morphological changes...... with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK severely impaired the pro-inflammatory response of MSCs to tumor CM (~80-99%, and 55...

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

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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.

The phocein homologue SmMOB3 is essential for vegetative cell fusion and sexual development in the filamentous ascomycete Sordaria macrospora.

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Bernhards, Yasmine; Pöggeler, Stefanie

2011-04-01

Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development.

Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter

International Nuclear Information System (INIS)

Michael, S.; Montag, M.; Dott, W.

2013-01-01

The objective of this study was to compare the toxicological effects of different source-related ambient PM10 samples in regard to their chemical composition. In this context we investigated airborne PM from different sites in Aachen, Germany. For the toxicological investigation human alveolar epithelial cells (A549) and murine macrophages (RAW264.7) were exposed from 0 to 96 h to increasing PM concentrations (0–100 μg/ml) followed by analyses of cell viability, pro-inflammatory and oxidative stress responses. The chemical analysis of these particles indicated the presence of 21 elements, water-soluble ions and PAHs. The toxicological investigations of the PM10 samples demonstrated a concentration- and time-dependent decrease in cell viability and an increase in pro-inflammatory and oxidative stress markers. -- Highlights: ► The study compares the toxicological effects of different source-related particles with regard to their chemical composition. ► The chemical characterization of the coarse particles revealed clear differences in elemental, TC and PAH composition. ► Equal mass concentrations of urban traffic and rural PM caused different toxicological responses. ► The observations confirm the hypothesis that particle composition, as well as origin, influence the PM-induced toxicity. -- The toxicological responses of lung epithelial cells and macrophages differ significantly after an exposure to equal mass concentrations of urban traffic and rural PM

Dimethyl sulfoxide-caused changes in pro- and anti-angiogenic factor levels could contribute to an anti-angiogenic response in HeLa cells.

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Şimşek, Ece; Aydemir, Esra Arslan; İmir, Nilüfer; Koçak, Orhan; Kuruoğlu, Aykut; Fışkın, Kayahan

2015-10-01

Dimethyl sulfoxide (DMSO) is widely used in biological research as a general solvent. While it has been previously demonstrated that DMSO possesses a wide range of pharmacological effects, there is no published work regarding the effects of DMSO on pro-angiogenic factor levels. This study was designed to investigate the possible effects of DMSO on the levels of three pro-angiogenic factors released from HeLa cells in vitro. Cells were treated with two different and previously determined concentrations of DMSO. The cytotoxic effects of DMSO concentrations on HeLa cells were determined via MTT. Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay. Changes in the pro-angiogenic levels in media were evaluated by Cayman's Substance P Enzyme Immunoassay ELISA kit. Vascular endothelial growth factor ELISA kit and interferon gamma ELISA kit for substance P, VEGF and IFNγ respectively. Changes in substance P levels were corrected by standard western blotting. Changes in VEGF and IFNγ levels were corrected both by western blot and real time PCR. Treatment with 1.4 μM DMSO caused a time-dependent inhibition of cell proliferation at 24, 48 and 72 h. 1.4 μM DMSO caused a significant reduction in VEGF levels at 72 h of incubation and sharp increases in IFNγ levels at both 48 and 72 h of incubation. According to real time PCR analyses, DMSO (1.4 μM) exhibited an inhibitory effect on VEGF but acted as an augmenter of IFNγ release on HeLa cells in vitro. This is the first report showing that the general solvent DMSO suppressed HeLa cell proliferation, decreased the levels of two pro-angiogenic factors (substance P and VEGF) and increased the release of an anti-angiogenic factor IFNγ in vitro. Copyright © 2015 Elsevier Ltd. All rights reserved.

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

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Osiris Marroquin Belaunzaran

Full Text Available HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA. HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272 and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM. HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules.HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

Science.gov (United States)

Marroquin Belaunzaran, Osiris; Kleber, Sascha; Schauer, Stefan; Hausmann, Martin; Nicholls, Flora; Van den Broek, Maries; Payeli, Sravan; Ciurea, Adrian; Milling, Simon; Stenner, Frank; Shaw, Jackie; Kollnberger, Simon; Bowness, Paul; Petrausch, Ulf; Renner, Christoph

2015-01-01

Objectives HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. Methods The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. Results HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. Conclusion HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders. PMID:26125554

Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

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Harpa Karadottir

2015-12-01

Full Text Available Mechanical ventilation (MV of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37. Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses.

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

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Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Li, Qiuping [Zhongshan Hospital of Fudan University, Shanghai 200032 (China); Yang, Zhiyuan; Wu, Guoqiang [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Sun, Shuhui [Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Gu, Jianxin [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Institutes of Biomedical Sciences of Fudan University, Shanghai 200032 (China); Wei, Yuanyan, E-mail: yywei@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Jiang, Jianhai, E-mail: jianhaijiang@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China)

2010-07-09

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

International Nuclear Information System (INIS)

Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Li, Qiuping; Yang, Zhiyuan; Wu, Guoqiang; Sun, Shuhui; Gu, Jianxin; Wei, Yuanyan; Jiang, Jianhai

2010-01-01

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

Diabetes and overexpression of proNGF cause retinal neurodegeneration via activation of RhoA pathway.

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Mohammed M H Al-Gayyar

Full Text Available Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotocin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75(NTR, cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75(NTR and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75(NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

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Kusumastuti, Yuni; Shibasaki, Yoshiaki; Hirohara, Shiho; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

2017-03-01

Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

The COG database: an updated version includes eukaryotes

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Sverdlov Alexander V

2003-09-01

Full Text Available Abstract Background The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. Results We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens, one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the

Human Langerhans Cells with Pro-inflammatory Features Relocate within Psoriasis Lesions

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Eidsmo, Liv; Martini, Elisa

2018-01-01

Psoriasis is a common skin disease that presents with well-demarcated patches of inflammation. Recurrent disease in fixed areas of the skin indicates a localized disease memory that is preserved in resolved lesions. In line with such concept, the involvement of tissue-resident immune cells in psoriasis pathology is increasingly appreciated. Langerhans cells (LCs) are perfectly placed to steer resident T cells and local tissue responses in psoriasis. Here, we present an overview of the current knowledge of LCs in human psoriasis, including findings that highlight pro-inflammatory features of LCs in psoriasis lesions. We also review the literature on conflicting data regarding LC localization and functionality in psoriasis. Our review highlights that further studies are needed to elucidate the molecular mechanisms that drive LCs functionality in inflammatory diseases. PMID:29520279

Pro-inflammatory Cytokines Are Involved in Fluoride-Induced Cytotoxic Potential in HeLa Cells.

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Wang, Hong-Wei; Zhou, Bian-Hua; Cao, Jian-Wen; Zhao, Jing; Zhao, Wen-Peng; Tan, Pan-Pan

2017-01-01

This study was designed to investigate the pro-inflammatory cytokines and their involvement in the cytotoxic potential of fluoride (F) in HeLa cells. HeLa cells were cultured with varying F concentrations (1-50 mg/L) for 48 h, and treatment effects were analyzed. The viability of HeLa cells was determined with a colorimetric method. The concentrations of IL-1β, IL-2, IL-6, and TNF-a in culture supernatant were measured through enzyme linked immunosorbent assay (ELISA). The mRNA expression levels of IL-1β, IL-2, IL-6 and TNF-a were subjected to transcript analysis and quantified through reverse transcription real-time PCR. Results showed that 10, 20 and 50 mg/L F significantly decreased the viability of HeLa cells incubated for 24 and 48 h. With their cytotoxic effect, the concentrations of IL-1β, IL-2, IL-6, and TNF-a decreased significantly in response to F, especially at 20 and 50 mg/L for 48 h. The mRNA expression levels of IL-1β, IL-2, IL-6, and TNF-a were downregulated at 50 mg/L F for 48 h. Therefore, F inhibited HeLa cell growth; as such, F could be used to alleviate the inhibition of pro-inflammatory cytokine expression.

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).

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Hobson, Neil; Deyholos, Michael K

2013-04-01

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

DEFF Research Database (Denmark)

Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils

2015-01-01

The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly......, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations....... and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate...

A molecular timescale of eukaryote evolution and the rise of complex multicellular life

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Hedges, S. Blair; Blair, Jaime E.; Venturi, Maria L.; Shoe, Jason L.

2004-01-01

BACKGROUND: The pattern and timing of the rise in complex multicellular life during Earth's history has not been established. Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree. Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time. RESULTS: Our phylogenetic analyses revealed that (i) animals are more closely related to fungi than to plants, (ii) red algae are closer to plants than to animals or fungi, (iii) choanoflagellates are closer to animals than to fungi or plants, (iv) diplomonads, euglenozoans, and alveolates each are basal to plants+animals+fungi, and (v) diplomonads are basal to other eukaryotes (including alveolates and euglenozoans). Divergence times were estimated from global and local clock methods using 20-188 proteins per node, with data treated separately (multigene) and concatenated (supergene). Different time estimation methods yielded similar results (within 5%): vertebrate-arthropod (964 million years ago, Ma), Cnidaria-Bilateria (1,298 Ma), Porifera-Eumetozoa (1,351 Ma), Pyrenomycetes-Plectomycetes (551 Ma), Candida-Saccharomyces (723 Ma), Hemiascomycetes-filamentous Ascomycota (982 Ma), Basidiomycota-Ascomycota (968 Ma), Mucorales-Basidiomycota (947 Ma), Fungi-Animalia (1,513 Ma), mosses-vascular plants (707 Ma), Chlorophyta-Tracheophyta (968 Ma), Rhodophyta-Chlorophyta+Embryophyta (1,428 Ma), Plantae-Animalia (1,609 Ma), Alveolata-plants+animals+fungi (1,973 Ma), Euglenozoa-plants+animals+fungi (1,961 Ma), and Giardia-plants+animals+fungi (2,309 Ma). By extrapolation, mitochondria arose approximately 2300-1800 Ma and plastids arose 1600-1500 Ma. Estimates of the maximum number of cell types of common ancestors, combined with divergence times, showed an increase from two cell types at 2500 Ma to

A molecular timescale of eukaryote evolution and the rise of complex multicellular life

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Venturi Maria L

2004-01-01

Full Text Available Abstract Background The pattern and timing of the rise in complex multicellular life during Earth's history has not been established. Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree. Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time. Results Our phylogenetic analyses revealed that (i animals are more closely related to fungi than to plants, (ii red algae are closer to plants than to animals or fungi, (iii choanoflagellates are closer to animals than to fungi or plants, (iv diplomonads, euglenozoans, and alveolates each are basal to plants+animals+fungi, and (v diplomonads are basal to other eukaryotes (including alveolates and euglenozoans. Divergence times were estimated from global and local clock methods using 20–188 proteins per node, with data treated separately (multigene and concatenated (supergene. Different time estimation methods yielded similar results (within 5%: vertebrate-arthropod (964 million years ago, Ma, Cnidaria-Bilateria (1,298 Ma, Porifera-Eumetozoa (1,351 Ma, Pyrenomycetes-Plectomycetes (551 Ma, Candida-Saccharomyces (723 Ma, Hemiascomycetes-filamentous Ascomycota (982 Ma, Basidiomycota-Ascomycota (968 Ma, Mucorales-Basidiomycota (947 Ma, Fungi-Animalia (1,513 Ma, mosses-vascular plants (707 Ma, Chlorophyta-Tracheophyta (968 Ma, Rhodophyta-Chlorophyta+Embryophyta (1,428 Ma, Plantae-Animalia (1,609 Ma, Alveolata-plants+animals+fungi (1,973 Ma, Euglenozoa-plants+animals+fungi (1,961 Ma, and Giardia-plants+animals+fungi (2,309 Ma. By extrapolation, mitochondria arose approximately 2300-1800 Ma and plastids arose 1600-1500 Ma. Estimates of the maximum number of cell types of common ancestors, combined with divergence times, showed an increase from two cell types at 2500 Ma to ~10

RNase MRP and the RNA processing cascade in the eukaryotic ancestor.

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Woodhams, Michael D; Stadler, Peter F; Penny, David; Collins, Lesley J

2007-02-08

Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor. We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution. We conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

Different polyamine pathways from bacteria have replaced eukaryotic spermidine biosynthesis in ciliates Tetrahymena thermophila and Paramecium tetaurelia.

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Li, Bin; Kim, Sok Ho; Zhang, Yang; Hanfrey, Colin C; Elliott, Katherine A; Ealick, Steven E; Michael, Anthony J

2015-09-01

The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase. © 2015 John Wiley & Sons Ltd.

[MiRNA system in unicellular eukaryotes and its evolutionary implications].

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Zhang, Yan-Qiong; Wen, Jian-Fan

2010-02-01

microRNAs (miRNAs) in higher multicellular eukaryotes have been extensively studied in recent years. Great progresses have also been achieved for miRNAs in unicellular eukaryotes. All these studies not only enrich our knowledge about the complex expression regulation system in diverse organisms, but also have evolutionary significance for understanding the origin of this system. In this review, Authors summarize the recent advance in the studies of miRNA in unicellular eukaryotes, including that on the most primitive unicellular eukaryote--Giardia. The origin and evolution of miRNA system is also discussed.

An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

Czech Academy of Sciences Publication Activity Database

Hronová, Vladislava; Valášek, Leoš

2017-01-01

Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 7.725, year: 2016

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

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Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-11-24

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

proBAMconvert: A Conversion Tool for proBAM/proBed.

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Olexiouk, Volodimir; Menschaert, Gerben

2017-07-07

The introduction of new standard formats, proBAM and proBed, improves the integration of genomics and proteomics information, thus aiding proteogenomics applications. These novel formats enable peptide spectrum matches (PSM) to be stored, inspected, and analyzed within the context of the genome. However, an easy-to-use and transparent tool to convert mass spectrometry identification files to these new formats is indispensable. proBAMconvert enables the conversion of common identification file formats (mzIdentML, mzTab, and pepXML) to proBAM/proBed using an intuitive interface. Furthermore, ProBAMconvert enables information to be output both at the PSM and peptide levels and has a command line interface next to the graphical user interface. Detailed documentation and a completely worked-out tutorial is available at http://probam.biobix.be .

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

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Sato, S.M.; Mains, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

1987-08-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

International Nuclear Information System (INIS)

Sato, S.M.; Mains, R.E.

1987-01-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment

Snapshot of the eukaryotic gene expression in muskoxen rumen--a metatranscriptomic approach.

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Meng Qi

Full Text Available BACKGROUND: Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus, with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6, GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. CONCLUSIONS/SIGNIFICANCE: The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes.

Snapshot of the Eukaryotic Gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach

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O'Toole, Nicholas; Barboza, Perry S.; Ungerfeld, Emilio; Leigh, Mary Beth; Selinger, L. Brent; Butler, Greg; Tsang, Adrian; McAllister, Tim A.; Forster, Robert J.

2011-01-01

Background Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. Methodology/Principal Findings In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. Conclusions/Significance The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes. PMID:21655220

Antioxidants inhibit SAA formation and pro-inflammatory cytokine release in a human cell model of alkaptonuria.

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Spreafico, Adriano; Millucci, Lia; Ghezzi, Lorenzo; Geminiani, Michela; Braconi, Daniela; Amato, Loredana; Chellini, Federico; Frediani, Bruno; Moretti, Elena; Collodel, Giulia; Bernardini, Giulia; Santucci, Annalisa

2013-09-01

Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease that currently lacks an appropriate therapy. Recently we provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis. The aim of the present work was to evaluate the use of antioxidants to inhibit SAA amyloid and pro-inflammatory cytokine release in AKU. We adopted a human chondrocytic cell AKU model to evaluate the anti-amyloid capacity of a set of antioxidants that had previously been shown to counteract ochronosis in a serum AKU model. Amyloid presence was evaluated by Congo red staining. Homogentisic acid-induced SAA production and pro-inflammatory cytokine release (overexpressed in AKU patients) were evaluated by ELISA and multiplex systems, respectively. Lipid peroxidation was evaluated by means of a fluorescence-based assay. Our AKU model allowed us to prove the efficacy of ascorbic acid combined with N-acetylcysteine, taurine, phytic acid and lipoic acid in significantly inhibiting SAA production, pro-inflammatory cytokine release and membrane lipid peroxidation. All the tested antioxidant compounds were able to reduce the production of amyloid and may be the basis for establishing new therapies for AKU amyloidosis.

Current Perspectives of Telomerase Structure and Function in Eukaryotes with Emerging Views on Telomerase in Human Parasites.

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Dey, Abhishek; Chakrabarti, Kausik

2018-01-24

Replicative capacity of a cell is strongly correlated with telomere length regulation. Aberrant lengthening or reduction in the length of telomeres can lead to health anomalies, such as cancer or premature aging. Telomerase is a master regulator for maintaining replicative potential in most eukaryotic cells. It does so by controlling telomere length at chromosome ends. Akin to cancer cells, most single-cell eukaryotic pathogens are highly proliferative and require persistent telomerase activity to maintain constant length of telomere and propagation within their host. Although telomerase is key to unlimited cellular proliferation in both cases, not much was known about the role of telomerase in human parasites (malaria, Trypanosoma , etc.) until recently. Since telomerase regulation is mediated via its own structural components, interactions with catalytic reverse transcriptase and several factors that can recruit and assemble telomerase to telomeres in a cell cycle-dependent manner, we compare and discuss here recent findings in telomerase biology in cancer, aging and parasitic diseases to give a broader perspective of telomerase function in human diseases.

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset.

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Victoria L Palmer

Full Text Available We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC, a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220(+CD43(+ and pre-B (B220(+CD43(- cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21-38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.

Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

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Mary M Melia

Full Text Available Signalling lymphocyte activation molecule (SLAM has been identified as an immune cell receptor for the morbilliviruses, measles (MV, canine distemper (CDV, rinderpest and peste des petits ruminants (PPRV viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4, also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF,for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

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Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

2009-12-01

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

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Ugarte-Berzal, Estefanía; Bailón, Elvira; Amigo-Jiménez, Irene; Albar, Juan Pablo; García-Marco, José A; García-Pardo, Angeles

2014-05-30

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities.

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Ray, Ann; Schwartz, Nika; de Souza Santos, Marcela; Zhang, Junmei; Orth, Kim; Salomon, Dor

2017-11-01

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense.

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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.

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis.

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G Hodge

Full Text Available Bronchiectasis (BE in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin and inflammatory (IFNγ and TNFα mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE.Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry.There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL.Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

New insights into the structural organization of eukaryotic and prokaryotic cytoskeletons using cryo-electron tomography

International Nuclear Information System (INIS)

Kuerner, Julia; Medalia, Ohad; Linaroudis, Alexandros A.; Baumeister, Wolfgang

2004-01-01

Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3-D) imaging at molecular resolution (<5 nm) with a close-to-life preservation of the specimen. In conjunction with pattern recognition techniques, it enables us to map the molecular landscape inside cells. The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells

Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

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Claire M Perks

2011-05-01

Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

Retracted: Effects of pro-inflammatory cytokines on mineralization potential of rat dental pulp stem cells

NARCIS (Netherlands)

Yang, X.; Walboomers, X.F.; Bian, Z.; Jansen, J.A.; Fan, M.

2011-01-01

The following article from the Journal of Tissue Engineering and Regenerative Medicine, 'Effects of Pro-inflammatory Cytokines on Mineralization Potential of Rat Dental Pulp Stem Cells' by Yang X, Walboomers XF, Bian Z, Jansen JA, Fan M, published online on 11 July 2011 in Wiley Online Library

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

OpenAIRE

Huerta, Carlos; Borek, Dominika; Machius, Mischa; Grishin, Nick V.; Zhang, Hong

2009-01-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata...

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

Science.gov (United States)

Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

2017-05-15

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

Co-chaperone BAG2 Determines the Pro-oncogenic Role of Cathepsin B in Triple-Negative Breast Cancer Cells

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Kyung-Min Yang

2017-12-01

Full Text Available Summary: Triple-negative breast cancer (TNBC is considered incurable with currently available treatments, highlighting the need for therapeutic targets and predictive biomarkers. Here, we report a unique role for Bcl-2-associated athanogene 2 (BAG2, which is significantly overexpressed in TNBC, in regulating the dual functions of cathepsin B as either a pro- or anti-oncogenic enzyme. Silencing BAG2 suppresses tumorigenesis and lung metastasis and induces apoptosis by increasing the intracellular mature form of cathepsin B, whereas BAG2 expression induces metastasis by blocking the auto-cleavage processing of pro-cathepsin B via interaction with the propeptide region. BAG2 regulates pro-cathepsin B/annexin II complex formation and facilitates the trafficking of pro-cathespin-B-containing TGN38-positive vesicles toward the cell periphery, leading to the secretion of pro-cathepsin B, which induces metastasis. Collectively, our results uncover BAG2 as a regulator of the oncogenic function of pro-cathepsin B and a potential diagnostic and therapeutic target that may reduce the burden of metastatic breast cancer. : The mechanisms controlling the pro- and anti-oncogenic roles of cathepsin B are unclear. Yang et al. find that BAG2 is a regulator of the dual functions of its client protein, CTSB, facilitating the progression of TNBC. Keywords: BAG2, cathepsin B, TNBC, tumorigenesis, metastasis, breast cancer, TGN38

Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

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Taparia, Shruti Sanjay; Khanna, Aparna

2016-10-01

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease

Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

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Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

2016-08-26

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pericellular activation of proMMP-7 (promatrilysin-1) through interaction with CD151.

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Shiomi, Takayuki; Inoki, Isao; Kataoka, Fumio; Ohtsuka, Takashi; Hashimoto, Gakuji; Nemori, Ryoichi; Okada, Yasunori

2005-12-01

Matrix metalloproteinase-7 (MMP-7) (also known as matrilysin-1) is secreted as a proenzyme (proMMP-7) and plays a key role in the degradation of various extracellular matrix (ECM) and non-ECM molecules after activation. To identify the binding proteins related to proMMP-7 activation, a human lung cDNA library was screened by yeast two-hybrid system using proMMP-7 as bait. We identified a candidate molecule CD151, which is a member of the transmembrane 4 superfamily. Complex formation of proMMP-7 with CD151 was demonstrated by immunoprecipitation of the molecules from CaR-1 cells, a human rectal carcinoma cell line, expressing both proMMP-7 and CD151, and CD151 stable transfectants incubated with proMMP-7. Yeast two-hybrid assays using deletion mutants of proMMP-7 and CD151 suggested an interaction between the propeptide of proMMP-7 and the COOH-terminal extracellular loop of CD151. The binding activity of (125)I-labeled proMMP-7 to CD151 on the cell membranes was shown with CD151 stable transfectants. Laser-scanning confocal microscopy demonstrated that proMMP-7 colocalizes with CD151 on the cell membranes of CD151 stable transfectants and CaR-1 cells. In situ zymography using crosslinked carboxymethylated transferrin, a substrate of MMP-7, demonstrated proteinase activity on and around CD151 stable transfectants and CaR-1 cells, while the activity was abolished by their treatment with MMP inhibitors, anti-MMP-7 antibody or anti-CD151 antibody. In human lung adenocarcinoma tissues, colocalization of MMP-7 and CD151 was demonstrated on the carcinoma cells. Metalloproteinase activity was present in these tissues and could be inhibited by antibodies to MMP-7 or CD151. These data demonstrate for the first time that proMMP-7 is captured and activated on the cell membranes through interaction with CD151, and suggest the possibility that similar to the MT1-MMP/MMP-2 system, MMP-7 is involved in the pericellular activation mechanism mediated by CD151, a crucial step in

How natural a kind is "eukaryote?".

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Doolittle, W Ford

2014-06-02

Systematics balances uneasily between realism and nominalism, uncommitted as to whether biological taxa are discoveries or inventions. If the former, they might be taken as natural kinds. I briefly review some philosophers' concepts of natural kinds and then argue that several of these apply well enough to "eukaryote." Although there are some sticky issues around genomic chimerism and when eukaryotes first appeared, if we allow for degrees in the naturalness of kinds, existing eukaryotes rank highly, higher than prokaryotes. Most biologists feel this intuitively: All I attempt to do here is provide some conceptual justification. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Apoptotic effects of antilymphocyte globulins on human pro-inflammatory CD4+CD28- T-cells.

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Christina Duftner

Full Text Available BACKGROUND: Pro-inflammatory, cytotoxic CD4(+CD28(- T-cells with known defects in apoptosis have been investigated as markers of premature immuno-senescence in various immune-mediated diseases. In this study we evaluated the influence of polyclonal antilymphocyte globulins (ATG-Fresenius, ATG-F on CD4(+CD28(- T-cells in vivo and in vitro. PRINCIPAL FINDINGS: Surface and intracellular three colour fluorescence activated cell sorting analyses of peripheral blood mononuclear cells from 16 consecutive transplant recipients and short-term cell lines were performed. In vivo, peripheral levels of CD3(+CD4(+CD28(- T-cells decreased from 3.7 ± 7.1% before to 0 ± 0% six hours after ATG-F application (P = 0.043 in 5 ATG-F treated but not in 11 control patients (2.9 ± 2.9% vs. 3.9 ± 3.0%. In vitro, ATG-F induced apoptosis even in CD4(+CD28(- T-cells, which was 4.3-times higher than in CD4(+CD28(+ T-cells. ATG-F evoked apoptosis was partially reversed by the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz-Val-Ala-Asp(OMe-fluoromethylketone (zVAD-fmk and prednisolon-21-hydrogensuccinate. ATG-F triggered CD25 expression and production of pro-inflammatory cytokines, and induced down-regulation of the type 1 chemokine receptors CXCR-3, CCR-5, CX3CR-1 and the central memory adhesion molecule CD62L predominately in CD4(+CD28(- T-cells. CONCLUSION: In summary, in vivo depletion of peripheral CD3(+CD4(+CD28(- T-cells by ATG-F in transplant recipients was paralleled in vitro by ATG-F induced apoptosis. CD25 expression and chemokine receptor down-regulation in CD4(+CD28(- T-cells only partly explain the underlying mechanism.

ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typhimurium Cells on a Stainless Steel Surface

Science.gov (United States)

Finn, Sarah; Händler, Kristian; Condell, Orla; Colgan, Aoife; Cooney, Shane; McClure, Peter; Amézquita, Aléjandro; Hinton, Jay C. D.

2013-01-01

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods. PMID:23666329

Loss of a Single Mcl-1 Allele Inhibits MYC-Driven Lymphomagenesis by Sensitizing Pro-B Cells to Apoptosis

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Stephanie Grabow

2016-03-01

Full Text Available MCL-1 is critical for progenitor cell survival during emergency hematopoiesis, but its role in sustaining cells undergoing transformation and in lymphomagenesis is only poorly understood. We investigated the importance of MCL-1 in the survival of B lymphoid progenitors undergoing MYC-driven transformation and its functional interactions with pro-apoptotic BIM and PUMA and the tumor suppressor p53 in lymphoma development. Loss of one Mcl-1 allele almost abrogated MYC-driven-lymphoma development owing to a reduction in lymphoma initiating pre-B cells. Although loss of the p53 target PUMA had minor impact, loss of one p53 allele substantially accelerated lymphoma development when MCL-1 was limiting, most likely because p53 loss also causes defects in non-apoptotic tumor suppressive processes. Remarkably, loss of BIM restored the survival of lymphoma initiating cells and rate of tumor development. Thus, MCL-1 has a major role in lymphoma initiating pro-B cells to oppose BIM, which is upregulated in response to oncogenic stress.

Intracerebroventricular Infusion of the (Pro)renin Receptor Antagonist PRO20 Attenuates Deoxycorticosterone Acetate-Salt–Induced Hypertension

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Li, Wencheng; Sullivan, Michelle N.; Zhang, Sheng; Worker, Caleb J.; Xiong, Zhenggang; Speth, Robert C.; Feng, Yumei

2016-01-01

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT1 receptor–dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. PMID:25421983

Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

Science.gov (United States)

Li, Wencheng; Sullivan, Michelle N; Zhang, Sheng; Worker, Caleb J; Xiong, Zhenggang; Speth, Robert C; Feng, Yumei

2015-02-01

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. © 2014 American Heart Association, Inc.

HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

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Santiago Guerrero

2015-01-01

Full Text Available Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1 uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

DEFF Research Database (Denmark)

Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

2013-01-01

BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

Complex multicellular functions at a unicellular eukaryote level: Learning, memory, and immunity.

Science.gov (United States)

Csaba, György

2017-06-01

According to experimental data, eukaryote unicellulars are able to learn, have immunity and memory. Learning is carried out in a very primitive form, and the memory is not neural but an epigenetic one. However, this epigenetic memory, which is well justified by the presence and manifestation of hormonal imprinting, is strong and permanent in the life of cell and also in its progenies. This memory is epigenetically executed by the alteration and fixation of methylation pattern of genes without changes in base sequences. The immunity of unicellulars is based on self/non-self discrimination, which leads to the destruction of non-self invaders and utilization of them as nourishment (by phagocytosis). The tools of learning, memory, and immunity of unicellulars are uniformly found in plasma membrane receptors, which formed under the effect of dynamic receptor pattern generation, suggested by Koch et al., and this is the basis of hormonal imprinting, by which the encounter between a chemical substance and the cell is specifically memorized. The receptors and imprinting are also used in the later steps of evolution up to mammals (including man) in each mentioned functions. This means that learning, memory, and immunity can be deduced to a unicellular eukaryote level.

Identification of a novel pro-inflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis

Science.gov (United States)

LAGGNER, Ute; DI MEGLIO, Paola; PERERA, Gayathri K.; HUNDHAUSEN, Christian; LACY, Katie E.; ALI, Niwa; SMITH, Catherine H.; HAYDAY, Adrian C.; NICKOLOFF, Brian J.; NESTLE, Frank O.

2011-01-01

γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is poorly characterized. In this study we show in vivo evidence that human blood contains a distinct subset of pro-inflammatory cutaneous lymphocyte antigen (CLA) and C-C chemokine receptor (CCR) 6 positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of pro-inflammatory mediators including IL-17A and activated keratinocytes in a TNF-α and IFN-γ dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared to healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, this data indicates redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human pro-inflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease. PMID:21813772

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

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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.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites [version 1; referees: 4 approved

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Olivier Hyrien

2016-08-01

Full Text Available DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs, the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC, they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Polyglutamine repeats are associated to specific sequence biases that are conserved among eukaryotes.

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Matteo Ramazzotti

Full Text Available Nine human neurodegenerative diseases, including Huntington's disease and several spinocerebellar ataxia, are associated to the aggregation of proteins comprising an extended tract of consecutive glutamine residues (polyQs once it exceeds a certain length threshold. This event is believed to be the consequence of the expansion of polyCAG codons during the replication process. This is in apparent contradiction with the fact that many polyQs-containing proteins remain soluble and are encoded by invariant genes in a number of eukaryotes. The latter suggests that polyQs expansion and/or aggregation might be counter-selected through a genetic and/or protein context. To identify this context, we designed a software that scrutinize entire proteomes in search for imperfect polyQs. The nature of residues flanking the polyQs and that of residues other than Gln within polyQs (insertions were assessed. We discovered strong amino acid residue biases robustly associated to polyQs in the 15 eukaryotic proteomes we examined, with an over-representation of Pro, Leu and His and an under-representation of Asp, Cys and Gly amino acid residues. These biases are conserved amongst unrelated proteins and are independent of specific functional classes. Our findings suggest that specific residues have been co-selected with polyQs during evolution. We discuss the possible selective pressures responsible of the observed biases.

Atypical mitochondrial inheritance patterns in eukaryotes.

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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.

Antioxidant, pro-oxidant and cytotoxic properties of parsley.

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Dorman, H J Damien; Lantto, Tiina A; Raasmaja, Atso; Hiltunen, Raimo

2011-06-01

Parsley (Petroselinum crispum) leaves were macerated with a mixture of methanol: water: acetic acid to produce a crude extract which was then defatted with (40°-60°) petrol. Antioxidant activity of the extract was evaluated using a battery of in vitro assays, viz., iron(iii) reduction, iron(ii) chelation and free radical scavenging assays. Evaluation of the pro-oxidant activity of the extract was based upon its effects upon DNA fragmentation and protein carbonylation. Cytotoxicity and apoptotic effects of the extract were determined in non-cancerous CV1-P fibroblast and cancerous A375 melanoma cells using MTT and LDH tests and caspase 3-like activity assay. The highest concentration, 2.0 mg ml(-1), decreased the viability of both cell lines, however, the cancerous melanoma cells were slightly susceptible to the effects. The observed cytotoxicity was not due to the caspase 3 activity. In conclusion, the toxicity might be explained by the pro-oxidative activity of components within the extract against proteins and/or DNA but it is not related to caspase 3-dependent apoptosis within cells.

Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host-pathogen interactions

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Laura eGomez-Valero

2011-10-01

Full Text Available Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic-like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

[Leu31, Pro34]neuropeptide Y

DEFF Research Database (Denmark)

Fuhlendorff, J; Gether, U; Aakerlund, L

1990-01-01

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do...... not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog......, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31...

Eukaryotes first: how could that be?

Science.gov (United States)

Mariscal, Carlos; Doolittle, W Ford

2015-09-26

In the half century since the formulation of the prokaryote : eukaryote dichotomy, many authors have proposed that the former evolved from something resembling the latter, in defiance of common (and possibly common sense) views. In such 'eukaryotes first' (EF) scenarios, the last universal common ancestor is imagined to have possessed significantly many of the complex characteristics of contemporary eukaryotes, as relics of an earlier 'progenotic' period or RNA world. Bacteria and Archaea thus must have lost these complex features secondarily, through 'streamlining'. If the canonical three-domain tree in which Archaea and Eukarya are sisters is accepted, EF entails that Bacteria and Archaea are convergently prokaryotic. We ask what this means and how it might be tested. © 2015 The Author(s).

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

Directory of Open Access Journals (Sweden)

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.

Origin and evolution of SINEs in eukaryotic genomes.

Science.gov (United States)

Kramerov, D A; Vassetzky, N S

2011-12-01

Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements.

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.

Pro-anorexia and pro-recovery photo sharing: a tale of two warring tribes.

Science.gov (United States)

Yom-Tov, Elad; Fernandez-Luque, Luis; Weber, Ingmar; Crain, Steven P

2012-11-07

There is widespread use of the Internet to promote anorexia as a lifestyle choice. Pro-anorexia content can be harmful for people affected or at risk of having anorexia. That movement is actively engaged in sharing photos on social networks such as Flickr. To study the characteristics of the online communities engaged in disseminating content that encourages eating disorders (known as "pro-anorexia") and to investigate if the posting of such content is discouraged by the posting of recovery-oriented content. The extraction of pro-anorexia and pro-recovery photographs from the photo sharing site Flickr pertaining to 242,710 photos from 491 users and analyzing four separate social networks therein. Pro-anorexia and pro-recovery communities interact to a much higher degree among themselves than what is expected from the distribution of contacts (only 59-72% of contacts but 74-83% of comments are made to members inside the community). Pro-recovery users employ similar words to those used by pro-anorexia users to describe their photographs, possibly in order to ensure that their content appears when pro-anorexia users search for images. Pro-anorexia users who are exposed to comments from the opposite camp are less likely to cease posting pro-anorexia photographs than those who do not receive such comments (46% versus 61%), and if they cease, they do so approximately three months later. Our observations show two highly active communities, where most interaction is within each community. However, the pro-recovery community takes steps to ensure that their content is visible to the pro-anorexia community, both by using textual descriptions of their photographs that are similar to those used by the pro-anorexia group and by commenting to pro-anorexia content. The latter activity is, however, counterproductive, as it entrenches pro-anorexia users in their stance. Our results highlight the nature of pro-anorexia and pro-recovery photo sharing and accentuate the need for

Pro-Anorexia and Pro-Recovery Photo Sharing: A Tale of Two Warring Tribes

Science.gov (United States)

Yom-Tov, Elad; Weber, Ingmar; Crain, Steven P

2012-01-01

Background There is widespread use of the Internet to promote anorexia as a lifestyle choice. Pro-anorexia content can be harmful for people affected or at risk of having anorexia. That movement is actively engaged in sharing photos on social networks such as Flickr. Objective To study the characteristics of the online communities engaged in disseminating content that encourages eating disorders (known as “pro-anorexia”) and to investigate if the posting of such content is discouraged by the posting of recovery-oriented content. Methods The extraction of pro-anorexia and pro-recovery photographs from the photo sharing site Flickr pertaining to 242,710 photos from 491 users and analyzing four separate social networks therein. Results Pro-anorexia and pro-recovery communities interact to a much higher degree among themselves than what is expected from the distribution of contacts (only 59-72% of contacts but 74-83% of comments are made to members inside the community). Pro-recovery users employ similar words to those used by pro-anorexia users to describe their photographs, possibly in order to ensure that their content appears when pro-anorexia users search for images. Pro-anorexia users who are exposed to comments from the opposite camp are less likely to cease posting pro-anorexia photographs than those who do not receive such comments (46% versus 61%), and if they cease, they do so approximately three months later. Our observations show two highly active communities, where most interaction is within each community. However, the pro-recovery community takes steps to ensure that their content is visible to the pro-anorexia community, both by using textual descriptions of their photographs that are similar to those used by the pro-anorexia group and by commenting to pro-anorexia content. The latter activity is, however, counterproductive, as it entrenches pro-anorexia users in their stance. Conclusions Our results highlight the nature of pro-anorexia and pro

Consistent mutational paths predict eukaryotic thermostability

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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.

Prediction about severity and outcome of sepsis by pro-atrial natriuretic peptide and pro-adrenomedullin.

Science.gov (United States)

Wang, Rui-lan; Kang, Fu-xin

2010-06-01

Measurement of biomarkers is a potential approach to early prediction of the risk of mortality in patients with sepsis. The aim of the present study was to evaluate the prognostic value of pro-atrial natriuretic peptide (pro-ANP) and pro-adrenomedullin (pro-ADM) levels in a cohort of medical intensive care patients and to compare it with that of other known biomarkers and physiological scores. Blood samples of 51 consecutive critically ill patients admitted to the intensive care unit and 53 age-matched healthy control people were evaluated in this prospective study. The prognostic value of pro-ANP and pro-ADM levels was compared with that of acute physiology and chronic health evaluation (APACHE) II scores and various biomarkers such as C-reactive protein, interleukin-6 and procalcitonin. Pro-ANP and pro-ADM were detected by a new sandwich immunoassay. On admission, 25 patients had systemic inflammatory response syndrome (SIRS), 12 sepsis, 9 severe sepsis and 5 septic shock. At that time, the median levels (ng/ml) of pro-ANP and pro-ADM were 87.22 and 0.34 respectively in patients with SIRS, 1533.30 and 2.23 in those with sepsis, 1098.73 and 4.57 in those with severe sepsis, and 1933.94 and 8.21 in those with septic shock. With the increasing severity of disease, the levels of pro-ANP and pro-ADM were gradually increased. On admission, the circulating levels of pro-ANP and pro-ADM in patients with sepsis, severe sepsis, or septic shock were significantly higher in non-survivors than in survivors (P less than 0.05). In a receiver operating characteristic curve analysis for the survival of patients with sepsis, the areas under the curve (AUCs) for pro-ANP and pro-ADM were 0.89 and 0.87 respectively, which was similar to the AUCs for procalcitonin and APACHE II scores. Pro-ANP and pro-ADM are valuable biomarkers for prediction of severity of septic patients.

Development of the Porosity Calculation Procedure using Pro/E and Pro/Tool kit

Energy Technology Data Exchange (ETDEWEB)

Lee, Sangyong; Park, Chaneok; Sohn, Jongjoo [Korea Power Engineering Company, Inc., Daejeon (Korea, Republic of)

2013-05-15

When the number of cells is greater than several hundreds, and/or, the complexity of the system is very high like the reactor system, there may be some problems that have to be solved for the successful customization. The first problem that comes with large number of mesh cells is the time to finish the calculation is too long. The second problem that comes with the complex system, which means that the data base of the solid model is too big, and prohibits this approach altogether. The reason for this is that simply the Pro/E cannot handle too complex geometry. For example, a desktop workstation that has a 3.3 GHz CPU and a Quadrol FX 5600 graphic card cannot handle a system that has more than 150000 solid parts because it takes too long time to regenerate model data and it takes to long time to manipulating graphic data by graphic processor. Another problem is identified with the Pro/Tool kit API routines. The maximum memory size of the array that can be handled by the API is 4 mega bytes. Therefore, a limited number of solid parts can be handled in one time. Some more efforts should be exercised to solve these problems to get a powerful porosity calculation procedure.

Development of the Porosity Calculation Procedure using Pro/E and Pro/Tool kit

International Nuclear Information System (INIS)

Lee, Sangyong; Park, Chaneok; Sohn, Jongjoo

2013-01-01

When the number of cells is greater than several hundreds, and/or, the complexity of the system is very high like the reactor system, there may be some problems that have to be solved for the successful customization. The first problem that comes with large number of mesh cells is the time to finish the calculation is too long. The second problem that comes with the complex system, which means that the data base of the solid model is too big, and prohibits this approach altogether. The reason for this is that simply the Pro/E cannot handle too complex geometry. For example, a desktop workstation that has a 3.3 GHz CPU and a Quadrol FX 5600 graphic card cannot handle a system that has more than 150000 solid parts because it takes too long time to regenerate model data and it takes to long time to manipulating graphic data by graphic processor. Another problem is identified with the Pro/Tool kit API routines. The maximum memory size of the array that can be handled by the API is 4 mega bytes. Therefore, a limited number of solid parts can be handled in one time. Some more efforts should be exercised to solve these problems to get a powerful porosity calculation procedure

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

International Nuclear Information System (INIS)

Yu, Lingling; Zhao, Yingmin; Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin; Gu, Jian; Yu, Duonan

2016-01-01

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

Energy Technology Data Exchange (ETDEWEB)

Yu, Lingling [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Zhao, Yingmin [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin [Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Gu, Jian [Department of Hematology, Yangzhou University School of Clinical Medicine, Yangzhou 225001 (China); Yu, Duonan, E-mail: duonan@yahoo.com [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Disease, Yangzhou 225001 (China); Institute of Comparative Medicine, Yangzhou University, Yangzhou 225001 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou 225001 (China)

2016-06-10

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.

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

Patterns of intron gain and conservation in eukaryotic genes

Directory of Open Access Journals (Sweden)

Wolf Yuri I

2007-10-01

Full Text Available Abstract Background: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. Results: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. Conclusion: We obtained robust estimates of the contribution of parallel gain to the observed

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells.

Science.gov (United States)

Kuhn-Nentwig, Lucia; Willems, Jean; Seebeck, Thomas; Shalaby, Tarek; Kaiser, Marcel; Nentwig, Wolfgang

2011-01-01

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-D: - and all-L: -cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of L: -cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

Science.gov (United States)

Moen, Siv H; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J; Sponaas, Anne-Marit; Standal, Therese

2016-09-01

Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1β, IL-6, CXCL10, and in the inhibition of HGF and TGFβ. Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Science.gov (United States)

Brosius, Jürgen

2014-01-01

Currently, the best scenario for earliest forms of life is based on RNA molecules as they have the proven ability to catalyze enzymatic reactions and harbor genetic information. Evolutionary principles valid today become apparent in such models already. Furthermore, many features of eukaryotic genome architecture might have their origins in an RNA or RNA/protein (RNP) world, including the onset of a further transition, when DNA replaced RNA as the genetic bookkeeper of the cell. Chromosome maintenance, splicing, and regulatory function via RNA may be deeply rooted in the RNA/RNP worlds. Mostly in eukaryotes, conversion from RNA to DNA is still ongoing, which greatly impacts the plasticity of extant genomes. Raw material for novel genes encoding protein or RNA, or parts of genes including regulatory elements that selection can act on, continues to enter the evolutionary lottery. PMID:25081515

Origin and evolution of the self-organizing cytoskeleton in the network of eukaryotic organelles.

Science.gov (United States)

Jékely, Gáspár

2014-09-02

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing "active gel," the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Similar pro-NT and pro-RLX2 levels after preeclampsia and after uncomplicated pregnancy

NARCIS (Netherlands)

Zoet, G. A.(Gerbrand); van Rijn, B. B.(Bas); Rehfeldt, M. (Miriam); Franx, A. (Arie); Maas, Angela H E M

2017-01-01

OBJECTIVE: Women are at increased risk of developing cardiovascular disease (CVD) after preeclampsia. Proneurotensin 1-117 (pro-NT) and prorelaxin 2 connecting peptide (pro-RLX2) have recently emerged as potential biomarkers for CVD risk in women. We assessed pro-NT and pro-RLX2 levels in women with

Similar pro-NT and pro-RLX2 levels after preeclampsia and after uncomplicated pregnancy

NARCIS (Netherlands)

Zoet, G. A.(Gerbrand); van Rijn, B. B.(Bas); Rehfeldt, M. (Miriam); Franx, A. (Arie); Maas, Angela H E M

2017-01-01

Objective Women are at increased risk of developing cardiovascular disease (CVD) after preeclampsia. Proneurotensin 1-117 (pro-NT) and prorelaxin 2 connecting peptide (pro-RLX2) have recently emerged as potential biomarkers for CVD risk in women. We assessed pro-NT and pro-RLX2 levels in women with

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

Science.gov (United States)

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

Science.gov (United States)

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tenocytes, pro-inflammatory cytokines and leukocytes: a relationship?

OpenAIRE

Al-Sadi, Onays; Schulze-Tanzil, Gundula; Kohl, Benjamin; Lohan, Anke; Lemke, Marion; Ertel, Wolfgang; John, Thilo

2012-01-01

Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures.

Basic fibroblast growth factor is pro-adipogenic in rat skeletal muscle progenitor clone, 2G11 cells.

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Nakano, Shin-ichi; Nakamura, Katsuyuki; Teramoto, Naomi; Yamanouchi, Keitaro; Nishihara, Masugi

2016-01-01

Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo. © 2015 Japanese Society of Animal Science.

Interaction of tRNA with Eukaryotic Ribosome

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Dmitri Graifer

2015-03-01

Full Text Available This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria.

Eelgrass Leaf Surface Microbiomes Are Locally Variable and Highly Correlated with Epibiotic Eukaryotes

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Mia M. Bengtsson

2017-07-01

Full Text Available Eelgrass (Zostera marina is a marine foundation species essential for coastal ecosystem services around the northern hemisphere. Like all macroscopic organisms, it possesses a microbiome (here defined as an associated prokaryotic community which may play critical roles in modulating the interaction of eelgrass with its environment. For example, its leaf surface microbiome could inhibit or attract eukaryotic epibionts which may overgrow the eelgrass leading to reduced primary productivity and subsequent eelgrass meadow decline. We used amplicon sequencing of the 16S and 18S rRNA genes of prokaryotes and eukaryotes to assess the leaf surface microbiome (prokaryotes as well as eukaryotic epibionts in- and outside lagoons on the German Baltic Sea coast. Prokaryote microbiomes varied substantially both between sites inside lagoons and between open coastal and lagoon sites. Water depth, leaf area and biofilm chlorophyll a concentration explained a large amount of variation in both prokaryotic and eukaryotic community composition. The prokaryotic microbiome and eukaryotic epibiont communities were highly correlated, and network analysis revealed disproportionate co-occurrence between a limited number of eukaryotic taxa and several bacterial taxa. This suggests that eelgrass leaf surfaces are home to a mosaic of microbiomes of several epibiotic eukaryotes, in addition to the microbiome of the eelgrass itself. Our findings thereby underline that eukaryotic diversity should be taken into account in order to explain prokaryotic microbiome assembly and dynamics in aquatic environments.

Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells

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BA Walter

2016-07-01

Full Text Available The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4 ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease.

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

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Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

Large variability of bathypelagic microbial eukaryotic communities across the world's oceans.

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Pernice, Massimo C; Giner, Caterina R; Logares, Ramiro; Perera-Bel, Júlia; Acinas, Silvia G; Duarte, Carlos M; Gasol, Josep M; Massana, Ramon

2016-04-01

In this work, we study the diversity of bathypelagic microbial eukaryotes (0.8-20 μm) in the global ocean. Seawater samples from 3000 to 4000 m depth from 27 stations in the Atlantic, Pacific and Indian Oceans were analyzed by pyrosequencing the V4 region of the 18S ribosomal DNA. The relative abundance of the most abundant operational taxonomic units agreed with the results of a parallel metagenomic analysis, suggesting limited PCR biases in the tag approach. Although rarefaction curves for single stations were seldom saturated, the global analysis of all sequences together suggested an adequate recovery of bathypelagic diversity. Community composition presented a large variability among samples, which was poorly explained by linear geographic distance. In fact, the similarity between communities was better explained by water mass composition (26% of the variability) and the ratio in cell abundance between prokaryotes and microbial eukaryotes (21%). Deep diversity appeared dominated by four taxonomic groups (Collodaria, Chrysophytes, Basidiomycota and MALV-II) appearing in different proportions in each sample. Novel diversity amounted to 1% of the pyrotags and was lower than expected. Our study represents an essential step in the investigation of bathypelagic microbial eukaryotes, indicating dominating taxonomic groups and suggesting idiosyncratic assemblages in distinct oceanic regions.

Metabolism in anoxic permeable sediments is dominated by eukaryotic dark fermentation

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Bourke, Michael F.; Marriott, Philip J.; Glud, Ronnie N.; Hasler-Sheetal, Harald; Kamalanathan, Manoj; Beardall, John; Greening, Chris; Cook, Perran L. M.

2017-01-01

Permeable sediments are common across continental shelves and are critical contributors to marine biogeochemical cycling. Organic matter in permeable sediments is dominated by microalgae, which as eukaryotes have different anaerobic metabolic pathways to bacteria and archaea. Here we present analyses of flow-through reactor experiments showing that dissolved inorganic carbon is produced predominantly as a result of anaerobic eukaryotic metabolic activity. In our experiments, anaerobic production of dissolved inorganic carbon was consistently accompanied by large dissolved H2 production rates, suggesting the presence of fermentation. The production of both dissolved inorganic carbon and H2 persisted following administration of broad spectrum bactericidal antibiotics, but ceased following treatment with metronidazole. Metronidazole inhibits the ferredoxin/hydrogenase pathway of fermentative eukaryotic H2 production, suggesting that pathway as the source of H2 and dissolved inorganic carbon production. Metabolomic analysis showed large increases in lipid production at the onset of anoxia, consistent with documented pathways of anoxic dark fermentation in microalgae. Cell counts revealed a predominance of microalgae in the sediments. H2 production was observed in dark anoxic cultures of diatoms (Fragilariopsis sp.) and a chlorophyte (Pyramimonas) isolated from the study site, substantiating the hypothesis that microalgae undertake fermentation. We conclude that microalgal dark fermentation could be an important energy-conserving pathway in permeable sediments.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

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Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

2017-06-02

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-reconstruction for eukaryotes from complex natural microbial communities.

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West, Patrick T; Probst, Alexander J; Grigoriev, Igor V; Thomas, Brian C; Banfield, Jillian F

2018-04-01

Microbial eukaryotes are integral components of natural microbial communities, and their inclusion is critical for many ecosystem studies, yet the majority of published metagenome analyses ignore eukaryotes. In order to include eukaryotes in environmental studies, we propose a method to recover eukaryotic genomes from complex metagenomic samples. A key step for genome recovery is separation of eukaryotic and prokaryotic fragments. We developed a k -mer-based strategy, EukRep, for eukaryotic sequence identification and applied it to environmental samples to show that it enables genome recovery, genome completeness evaluation, and prediction of metabolic potential. We used this approach to test the effect of addition of organic carbon on a geyser-associated microbial community and detected a substantial change of the community metabolism, with selection against almost all candidate phyla bacteria and archaea and for eukaryotes. Near complete genomes were reconstructed for three fungi placed within the Eurotiomycetes and an arthropod. While carbon fixation and sulfur oxidation were important functions in the geyser community prior to carbon addition, the organic carbon-impacted community showed enrichment for secreted proteases, secreted lipases, cellulose targeting CAZymes, and methanol oxidation. We demonstrate the broader utility of EukRep by reconstructing and evaluating relatively high-quality fungal, protist, and rotifer genomes from complex environmental samples. This approach opens the way for cultivation-independent analyses of whole microbial communities. © 2018 West et al.; Published by Cold Spring Harbor Laboratory Press.

Expression and nutritional regulation of the (pro)renin receptor in rat visceral adipose tissue.

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Achard, V; Tassistro, V; Boullu-Ciocca, S; Grino, M

2011-12-01

Early life nutritional environment plays an important role in the development of visceral adipose tissue and interacts with nutritional regulations in adulthood, leading to metabolic dysregulations. We hypothesized that the renin-angiotensin system may play a role in the programming-induced development of visceral adipose tissue. We studied, using a model of programming of overweight and glucose intolerance, obtained by post-natal overfeeding with consecutive highfat diet, the status of plasma renin activity and mesenteric adipose renin-angiotensin system, including the recently identified (pro)renin receptor, in adult rats. Post-natal overfeeding or high-fat feeding lead to overweight with increased visceral fat mass and adipocytes surface. When both paradigms were associated, adipocytes surface showed a disproportionate increase. A strong immunoreactivity for (pro)renin receptor was found in stromal cells. Plasma renin activity increased in programmed animals whereas (pro)renin receptor expressing cells density was stimulated by high-fat diet. There was a positive, linear relationship between plasma renin activity and (pro)renin receptor expressing cells density and adipocytes surface. Our experiments demonstrate that association of post-natal overfeeding and high-fat diet increased plasma renin activity and adipose (pro)renin receptor expression. Such phenomenon could explain, at least in part, the associated disproportionate adipocyte hypertrophy and its accompanying increased glucose intolerance.

Cell-Nonautonomous Mechanisms Underlying Cellular and Organismal Aging.

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