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Sample records for recombinant cytosolic domains

  1. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  2. SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains.

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    Joshua Holcomb

    Full Text Available The Nogo-B receptor (NgBR is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs. Small Angle X-ray Scattering (SAXS analysis reveals the radius of gyration (Rg of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

  3. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    by recombinant technics to examine the relation between the TKl gene and the TKl protein. In the second part of this investigation a direct expression system for human TKl in E.coli was developed to produce a source of high amounts of TKl, to be able to examine the structure of TKl. The resulting recombinant TKl...... cells and that this modification can not be performed in E.coli....... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  4. Recombinant spider silk genetically functionalized with affinity domains.

    Science.gov (United States)

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  5. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

  6. Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1

    Directory of Open Access Journals (Sweden)

    Edavana VK

    2011-11-01

    Full Text Available Vineetha Koroth Edavana1, Xinfeng Yu1, Ishwori B Dhakal1, Suzanne Williams1, Baitang Ning2, Ian T Cook3, David Caldwell1, Charles N Falany3, Susan Kadlubar11Division of Medical Genetics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA; 2Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA; 3Department of Pharmacology, University of Alabama, Birmingham, AL, USAAbstract: Fulvestrant (Faslodex™ is a pure antiestrogen that is approved to treat hormone receptor-positive metastatic breast cancer in postmenopausal women. Previous studies have demonstrated that fulvestrant metabolism in humans involves cytochromes P450 and UDP-glucuronosyltransferases (UGTs. To date, fulvestrant sulfation has not been characterized. This study examined fulvestrant sulfation with nine recombinant sulfotransferases and found that only SULT1A1 and SULT1E1 displayed catalytic activity toward this substrate, with Km of 4.2 ± 0.99 and 0.2 ± 0.16 µM, respectively. In vitro assays of 104 human liver cytosols revealed marked individual variability that was highly correlated with β-naphthol sulfation (SULT1A1 diagnostic substrate; r = 0.98, P < 0.0001, but not with 17ß-estradiol sulfation (SULT1E1 diagnostic substrate; r = 0.16, P = 0.10. Fulvestrant sulfation was correlated with both SULT1A1*1/2 genotype (P value = 0.023 and copy number (P < 0.0001. These studies suggest that factors influencing SULT1A1/1E1 tissue expression and/or enzymatic activity could influence the efficacy of fulvestrant therapy.Keywords: fulvestrant, sulfotransferase, genotype, copy number

  7. Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.

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    Catherine J Pink

    Full Text Available In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in

  8. 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

  9. Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1

    DEFF Research Database (Denmark)

    Ellis, April L; Pan, Wensheng; Yang, Guang

    2010-01-01

    BACKGROUND: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human...... perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. RESULTS: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess...... glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary...

  10. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

    International Nuclear Information System (INIS)

    Panaviene, Zivile; Nagy, Peter D.

    2003-01-01

    RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

  11. Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

    Science.gov (United States)

    Machida, Kazuya; Liu, Bernard

    2017-01-01

    Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

  12. The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer.

    Science.gov (United States)

    Zimmermann, Kerstin; Eells, Rebecca; Heinrich, Frank; Rintoul, Stefanie; Josey, Brian; Shekhar, Prabhanshu; Lösche, Mathias; Stern, Lawrence J

    2017-10-27

    Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ cyt ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ cyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ cyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ cyt to the bilayers in dynamic equilibrium: one in which ζ cyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ cyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

  13. SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase.

    Science.gov (United States)

    Banerjee, Moumita; Duan, Qiming; Xie, Zijian

    2015-01-01

    Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.

  14. SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase.

    Directory of Open Access Journals (Sweden)

    Moumita Banerjee

    Full Text Available Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2 of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.

  15. Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

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    Adam Bajinting

    2017-06-01

    Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

  16. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    International Nuclear Information System (INIS)

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-01-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination

  17. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas; Wong, Aloysius Tze; Gehring, Christoph A

    2015-01-01

    KUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly

  18. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  19. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  20. The correlation between the length of repetitive domain and mechanical properties of the recombinant flagelliform spidroin

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    Xue Li

    2017-03-01

    Full Text Available Spider silk is an attractive biopolymer with numerous potential applications due to its remarkable characteristics. Among the six categories of spider silks, flagelliform (Flag spider silk possesses longer and more repetitive core domains than others, therefore performing the highest extensibility. To investigate the correlation between the recombinant spidroin size and the synthetic fiber properties, four recombinant proteins with different sizes [N-Scn-C (n=1-4] were constructed and expressed using IMPACT system. Subsequently, different recombinant spidroins were spun into fibers through wet-spinning via a custom-made continuous post-drawing device. Mechanical tests of the synthetic fibers with four parameters (maximum stress, maximum extension, Young's modulus and toughness demonstrated that the extensibility of the fibers showed a positive correlation with spidroin size, consequently resulting in the extensibility of N-Sc4-C fiber ranked the highest (58.76% among four fibers. Raman data revealed the relationship between secondary structure content and mechanical properties. The data here provide a deeper insight into the relationship between the function and structure of Flag silk for future design of artificial fibers.

  1. Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

    International Nuclear Information System (INIS)

    Wilhelm, O.G.; Jaskunas, S.R.; Vlahos, C.J.; Bang, N.U.

    1990-01-01

    The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

  2. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  3. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Science.gov (United States)

    Dioletis, Evangelos; Dingley, Andrew J; Driscoll, Paul C

    2013-01-01

    Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics

  4. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  5. Recombinant disintegrin domain of ADAM15 inhibits the proliferation and migration of Bel-7402 cells

    International Nuclear Information System (INIS)

    Hou, Y.; Chu, M.; Du, F.F.; Lei, J.Y.; Chen, Y.; Zhu, R.Y.; Gong, X.H.; Ma, X.; Jin, J.

    2013-01-01

    Highlights: •rhddADAM15 inhibited the proliferation and migration of Bel-7402 cells. •rhddADAM15 inhibited growth and metastasis of Bel-7402 cells in zebrafish xenograft. •rhddADAM15 induced apoptosis in Bel-7402 cells and somatic cells of zebrafish. •Cell-cycle in Bel-7402 cells showed a partial G 2 /S arrest. •Activity of caspases 8, 9 and 3 was increased in rhddADAM15-treated Bel-7402 cells. -- Abstract: ADAM15 (A Disintegrin And Metalloproteinase 15), a transmembrane protein containing seven domains, interacts with some integrins via its disintegrin domain and overexpresses in many solid tumors. In this study, the effect of the recombinant human disintegrin domain (rhddADAM15) on the proliferation and migration of Bel-7402 cells was evaluated in vitro and in vivo in zebrafish xenografts. rhddADAM15 (4 μM) severely inhibited the proliferation and migration of Bel-7402 cells, inducing a partial G 2 /S arrest and morphological nucleus changes of apoptosis. Moreover, the activity of caspases 8, 9 and 3 in Bel-7402 cells was increased. In addition, the zebrafish was used as a model for apoptosis-induction and tumor-xenograft. rhddADAM15 (1 pM) inhibited the growth and metastasis of Bel-7402 cell xenografts in zebrafish and a lower concentration (0.1 pM) induced severe apoptosis in the somatic cells of zebrafish. In conclusion, our data identified rhddADAM15 as a potent inhibitor of tumor growth and metastasis, making it a promising tool for use in anticancer treatment

  6. Recombinant disintegrin domain of ADAM15 inhibits the proliferation and migration of Bel-7402 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Y. [Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Rd., Wuxi, Jiangsu 214122 (China); Chu, M. [Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Medicine, Jiangnan University, 1800 Lihu Rd., Wuxi, Jiangsu 214122 (China); Du, F.F.; Lei, J.Y.; Chen, Y.; Zhu, R.Y.; Gong, X.H.; Ma, X. [Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Rd., Wuxi, Jiangsu 214122 (China); Jin, J., E-mail: jinjian31@126.com [Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Rd., Wuxi, Jiangsu 214122 (China)

    2013-06-14

    Highlights: •rhddADAM15 inhibited the proliferation and migration of Bel-7402 cells. •rhddADAM15 inhibited growth and metastasis of Bel-7402 cells in zebrafish xenograft. •rhddADAM15 induced apoptosis in Bel-7402 cells and somatic cells of zebrafish. •Cell-cycle in Bel-7402 cells showed a partial G{sub 2}/S arrest. •Activity of caspases 8, 9 and 3 was increased in rhddADAM15-treated Bel-7402 cells. -- Abstract: ADAM15 (A Disintegrin And Metalloproteinase 15), a transmembrane protein containing seven domains, interacts with some integrins via its disintegrin domain and overexpresses in many solid tumors. In this study, the effect of the recombinant human disintegrin domain (rhddADAM15) on the proliferation and migration of Bel-7402 cells was evaluated in vitro and in vivo in zebrafish xenografts. rhddADAM15 (4 μM) severely inhibited the proliferation and migration of Bel-7402 cells, inducing a partial G{sub 2}/S arrest and morphological nucleus changes of apoptosis. Moreover, the activity of caspases 8, 9 and 3 in Bel-7402 cells was increased. In addition, the zebrafish was used as a model for apoptosis-induction and tumor-xenograft. rhddADAM15 (1 pM) inhibited the growth and metastasis of Bel-7402 cell xenografts in zebrafish and a lower concentration (0.1 pM) induced severe apoptosis in the somatic cells of zebrafish. In conclusion, our data identified rhddADAM15 as a potent inhibitor of tumor growth and metastasis, making it a promising tool for use in anticancer treatment.

  7. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  8. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

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    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  9. In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences

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    Takashi Irie

    2012-01-01

    Full Text Available The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.

  10. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    Science.gov (United States)

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Hansen, Line V; Jørgensen, Thomas J D

    2007-01-01

    The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor...... as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant...... chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system...

  12. An Investigation of Immunogenicity of Chitosan-Based Botulinum Neurotoxin E Binding Domain Recombinant Candidate Vaccine via Mucosal Route

    Directory of Open Access Journals (Sweden)

    Mohammad Javad Bagheripour

    2017-01-01

    Full Text Available Background and Objectives: Botulism syndrome is caused by serotypes A-G of neurotoxins of Clostridium genus. Neurotoxin binding domain is an appropriate vaccine candidate due to its immunogenic activity. In this study, the immunogenicity of chitosan-based botulinum neurotoxin E binding domain recombinant candidate vaccine was investigated via mucosal route of administration. Methods: In this experimental study, chitosan nanoparticles containing rBoNT/E protein were synthesized by ionic gelation method and were administered orally and intranasally to mice. After each administration, IgG antibody titer was measured by ELISA method. Finally, all groups were challenged with active botulinum neurotoxin type E. Data were analyzed using Duncan and repeated ANOVA tests. The significance level was considered as p0.05, even intranasal route reduced the immunogenicity.

  13. Lewis Base Passivation of Hybrid Halide Perovskites Slows Electron-Hole Recombination: Time-Domain Ab Initio Analysis.

    Science.gov (United States)

    Liu, Lihong; Fang, Wei-Hai; Long, Run; Prezhdo, Oleg V

    2018-03-01

    Nonradiative electron-hole recombination plays a key role in determining photon conversion efficiencies in solar cells. Experiments demonstrate significant reduction in the recombination rate upon passivation of methylammonium lead iodide perovskite with Lewis base molecules. Using nonadiabatic molecular dynamics combined with time-domain density functional theory, we find that the nonradiative charge recombination is decelerated by an order of magnitude upon adsorption of the molecules. Thiophene acts by the traditional passivation mechanism, forcing electron density away from the surface. In contrast, pyridine localizes the electron at the surface while leaving it energetically near the conduction band edge. This is because pyridine creates a stronger coordinative bond with a lead atom of the perovskite and has a lower energy unoccupied orbital compared with thiophene due to the more electronegative nitrogen atom relative to thiophene's sulfur. Both molecules reduce two-fold the nonadiabatic coupling and electronic coherence time. A broad range of vibrational modes couple to the electronic subsystem, arising from inorganic and organic components. The simulations reveal the atomistic mechanisms underlying the enhancement of the excited-state lifetime achieved by the perovskite passivation, rationalize the experimental results, and advance our understanding of charge-phonon dynamics in perovskite solar cells.

  14. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

    Science.gov (United States)

    Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

    2013-06-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

  15. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    International Nuclear Information System (INIS)

    Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Danielsen, Mark

    2005-01-01

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

  16. 1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

    International Nuclear Information System (INIS)

    Byeon, I.J.L.; Llinas, M.; Kelley, R.F.

    1989-01-01

    The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1 H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu 46 , which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant K a ∼ 11.9 mM -1 . The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr 36 and, within the kringle inner loop, Trp 62 , His 64 , Trp 72 , and Tyr 74 . Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2 H 2 O for full deuteration in the presence of L-lysine at 37 degree C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1 H 2 O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His 48a imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5

  17. Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

    Science.gov (United States)

    Wang, Chuanwen; Liu, Liping; Zhang, Zhen; Yan, Zhengui; Yu, Cuilian; Shao, Mingxu; Jiang, Xiaodong; Chi, Shanshan; Wei, Kai; Zhu, Ruiliang

    2015-10-01

    Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

    Science.gov (United States)

    Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

    2013-01-01

    A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

  19. Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

    Science.gov (United States)

    Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

    2015-08-20

    For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    Science.gov (United States)

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

  1. Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

    Directory of Open Access Journals (Sweden)

    Dorra Zouari Ayadi

    2007-01-01

    Full Text Available We already reported the use of a long synthetic signal peptide (LSSP to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b. This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

  2. Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

    Directory of Open Access Journals (Sweden)

    Federico Cividini

    Full Text Available IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

  3. Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD

    Directory of Open Access Journals (Sweden)

    Moon Yuseok

    2009-01-01

    Full Text Available Abstract Background ATP binding cassette (ABC transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA, lipase ABC transporter domains (LARDs were designed for the secretion of fusion proteins. Results The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD to three types of proteins; green fluorescent protein (GFP, epidermal growth factor (EGF and cytoplasmic transduction peptide (CTP. These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant. Conclusion The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.

  4. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP71-100 generates cAMP in vitro.

  5. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  6. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  7. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

    Science.gov (United States)

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

    2014-01-01

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

  8. Antibody levels to recombinant VAR2CSA domains vary with Plasmodium falciparum parasitaemia, gestational age, and gravidity, but do not predict pregnancy outcomes.

    Science.gov (United States)

    Fried, Michal; Kurtis, Jonathan D; Swihart, Bruce; Morrison, Robert; Pond-Tor, Sunthorn; Barry, Amadou; Sidibe, Youssoufa; Keita, Sekouba; Mahamar, Almahamoudou; Andemel, Naissem; Attaher, Oumar; Dembele, Adama B; Cisse, Kadidia B; Diarra, Bacary S; Kanoute, Moussa B; Narum, David L; Dicko, Alassane; Duffy, Patrick E

    2018-03-09

    Maternal malaria is a tropical scourge associated with poor pregnancy outcomes. Women become resistant to Plasmodium falciparum pregnancy malaria as they acquire antibodies to the variant surface antigen VAR2CSA, a leading vaccine candidate. Because malaria infection may increase VAR2CSA antibody levels and thereby confound analyses of immune protection, gravidity-dependent changes in antibody levels during and after infection, and the effect of VAR2CSA antibodies on pregnancy outcomes were evaluated. Pregnant women enrolled in a longitudinal cohort study of mother-infant pairs in Ouelessebougou, Mali provided plasma samples at enrollment, gestational week 30-32, and delivery. Antibody levels to VAR2CSA domains were measured using a multiplex bead-based assay. Antibody levels to VAR2CSA were higher in multigravidae than primigravidae. Malaria infection was associated with increased antibody levels to VAR2CSA domains. In primigravidae but not in secundigravidae or multigravidae, antibodies levels sharply declined after an infection. A relationship between any VAR2CSA antibody specificity and protection from adverse pregnancy outcomes was not detected. During malaria infection, primigravidae acquire short-lived antibodies. The lack of an association between VAR2CSA domain antibody reactivity and improved pregnancy outcomes suggests that the recombinant proteins may not present native epitopes targeted by protective antibodies.

  9. Recombinant epidermal growth factor-like domain-1 from coagulation factor VII functionalized iron oxide nanoparticles for targeted glioma magnetic resonance imaging.

    Science.gov (United States)

    Liu, Heng; Chen, Xiao; Xue, Wei; Chu, Chengchao; Liu, Yu; Tong, Haipeng; Du, Xuesong; Xie, Tian; Liu, Gang; Zhang, Weiguo

    The highly infiltrative and invasive nature of glioma cells often leads to blurred tumor margins, resulting in incomplete tumor resection and tumor recurrence. Accurate detection and precise delineation of glioma help in preoperative delineation, surgical planning and survival prediction. In this study, recombinant epidermal growth factor-like domain-1, derived from human coagulation factor VII, was conjugated to iron oxide nanoparticles (IONPs) for targeted glioma magnetic resonance (MR) imaging. The synthesized EGF1-EGFP-IONPs exhibited excellent targeting ability toward tissue factor (TF)-positive U87MG cells and human umbilical vein endothelial cells in vitro, and demonstrated persistent and efficient MR contrast enhancement up to 12 h for preclinical glioma models with high targeting specificity in vivo. They hold great potential for clinical translation and developing targeted theranostics against brain glioma.

  10. The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag

    Directory of Open Access Journals (Sweden)

    Selma Djender

    2014-04-01

    Full Text Available We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.

  11. Neutralizing Antibody Response and Efficacy of Novel Recombinant Tetravalent Dengue DNA Vaccine Comprising Envelope Domain III in Mice

    Directory of Open Access Journals (Sweden)

    Ajit Kulkarni

    2017-03-01

    Full Text Available Background: Dengue is a global arboviral threat to humans; causing 390 million infections per year. The availability of safe and effective tetravalent dengue vaccine is a global requirement to prevent epidemics, morbidity, and mortality associated with it. Methods: Five experimental groups (6 mice per group each of 5-week-old BALB/c mice were immunized with vaccine and placebo (empty plasmid (100 µg, i.m. on days 0, 14 and 28. Among these, four groups (one group per serotype of each were subsequently challenged 3 weeks after the last boost with dengue virus (DENV serotypes 1-4 (100 LD50, 20 µl intracerebrally to determine vaccine efficacy. The fifth group of each was used as a control. The PBS immunized group was used as mock control. Serum samples were collected before and after subsequent immunizations. EDIII fusion protein expression was determined by Western blot. Total protein concentration was measured by Bradford assay. Neutralizing antibodies were assessed by TCID50-CPE inhibition assay. Statistical analysis was performed using Stata/IC 10.1 software for Windows. One-way repeated measures ANOVA and Mann-Whitney test were used for neutralizing antibody analysis and vaccine efficacy, respectively. Results: The recombinant EDIII fusion protein was expressed adequately in transfected 293T cells. Total protein concentration was almost 3 times more than the control. Vaccine candidate induced neutralizing antibodies against all four DENV serotypes with a notable increase after subsequent boosters. Vaccine efficacy was 83.3% (DENV-1, -3, -4 and 50% (DENV-2. Conclusion: Our results suggest that vaccine is immunogenic and protective; however, further studies are required to improve the immunogenicity particularly against DENV-2.

  12. Vaccination with Recombinant Non-transmembrane Domain of Protein Mannosyltransferase 4 Improves Survival during Murine Disseminated Candidiasis.

    Science.gov (United States)

    Wang, Li; Yan, Lan; Li, Xing Xing; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2015-01-01

    Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.

  13. Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro.

    Science.gov (United States)

    Yan, Xiaocai; Ma, Jun; Zheng, Jin; Lai, Baochang; Geng, Yiping; Wang, Yili; Si, Lüsheng

    2002-07-01

    To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

  14. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    remobilisation from ageing plant parts. Thus, GS is highly involved in determining crop yield and NUE. The major objective of this PhD project was to investigate the NUE properties of transgenic barley designed to constitutively overexpress a GS1 isogene (HvGS1.1). These transgenic lines exhibited an increased...... for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root...... fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  15. A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus.

    Science.gov (United States)

    Martina, Byron E E; van den Doel, Petra; Koraka, Penelope; van Amerongen, Geert; Spohn, Gunther; Haagmans, Bart L; Provacia, Lisette B V; Osterhaus, Albert D M E; Rimmelzwaan, Guus F

    2011-04-26

    West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.

  16. A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus.

    Directory of Open Access Journals (Sweden)

    Byron E E Martina

    Full Text Available West Nile virus (WNV continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5 TCID(50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.

  17. Cytosolic cholesterol ester hydrolase in adrenal cortex

    OpenAIRE

    Tocher, Douglas R.

    1983-01-01

    Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

  18. Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

    Science.gov (United States)

    Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

    2015-12-01

    Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

  19. Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

    Science.gov (United States)

    Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

    1992-11-01

    The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

  20. Redox characteristics of the eukaryotic cytosol

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Winther, Jakob R

    2007-01-01

    The eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species...... (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model...... organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist...

  1. Recombinant Programming

    OpenAIRE

    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

    2004-01-01

    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

  2. Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

    Science.gov (United States)

    Hernández Torres, Jorge; Papandreou, Nikolaos; Chomilier, Jacques

    2009-05-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

  3. Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

    Science.gov (United States)

    Papandreou, Nikolaos; Chomilier, Jacques

    2008-01-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

  4. Prediction of human pharmacokinetics of activated recombinant factor VII and B-domain truncated factor VIII from animal population pharmacokinetic models of haemophilia

    DEFF Research Database (Denmark)

    Larsen, Malte Selch; Juul, Rasmus Vestergaard; Groth, Andreas Velsing

    2018-01-01

    activated factor VII (rFVIIa) and recombinant factor VIII (rFVIII) in several experimental animal models using population PK modelling, and apply a simulation-based approach to evaluate how well the developed animal population PK models predict human PK. PK models were developed for rFVIIa and r...

  5. GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Bohmdorfer, G.; Schleiffer, A.; Brunmeir, R.; Ferscha, S.; Nizhynska, V.; Kozák, Jaroslav; Angelis, Karel; Kreil, D. P.; Schweizer, D.

    2011-01-01

    Roč. 67, č. 3 (2011), s. 420-433 ISSN 0960-7412 R&D Projects: GA MŠk 1M0505; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : structural maintenance of chromosomes * DNA repair * somatic homologous recombination Subject RIV: EI - Biotechnology ; Bionics Impact factor: 6.160, year: 2011

  6. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  7. Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications

    Science.gov (United States)

    2013-01-01

    Background Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Results Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other

  8. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra

    2013-01-01

    Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54...... and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage...

  9. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

    Directory of Open Access Journals (Sweden)

    Corentin Claeys Bouuaert

    2017-05-01

    Full Text Available Mlh1-Mlh3 (MutLγ is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ and, surprisingly, single-stranded DNA (ssDNA, which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced

  10. Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain

    NARCIS (Netherlands)

    van Wijnen, M.; Stam, J. G.; Chang, G. T.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

    1998-01-01

    Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain

  11. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  12. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  13. Cytosolic delivery of materials with endosome-disrupting colloids

    Science.gov (United States)

    Helms, Brett A.; Bayles, Andrea R.

    2016-03-15

    A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

  14. Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

    Science.gov (United States)

    Mazars, C; Thion, L; Thuleau, P; Graziana, A; Knight, M R; Moreau, M; Ranjeva, R

    1997-11-01

    Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

  15. The pharmacokinetics of a B-domain truncated recombinant factor VIII, turoctocog alfa (NovoEight®), in patients with hemophilia A.

    Science.gov (United States)

    Jiménez-Yuste, V; Lejniece, S; Klamroth, R; Suzuki, T; Santagostino, E; Karim, F A; Saugstrup, T; Møss, J

    2015-03-01

    Turoctocog alfa (NovoEight(®)) is a human recombinant coagulation factor VIII (rFVIII) for the treatment of patients with hemophilia A. To evaluate the pharmacokinetics of turoctocog alfa in all age groups across clinical trials. Data from previously treated patients with severe hemophilia A (FVIII activity level of ≤ 1%) with no history of FVIII inhibitors, in a non-bleeding state, were included. The pharmacokinetics were assessed following a wash-out period and a subsequent single intravenous 50 IU kg(-1) dose of turoctocog alfa. Blood was sampled during a 48-h period postdose. Standard pharmacokinetic (PK) parameters were estimated on the basis of plasma FVIII activity vs. time (PK profiles) with non-compartmental methods. Furthermore, a population PK analysis was conducted. Data from 76 patients (aged 1-60 years) enrolled globally across six clinical trials were included, totaling 105 turoctocog alfa PK profiles. Single-dose PK results 3-6 months after the first dose of turoctocog alfa were comparable with the results obtained after the first dose. Similar PK characteristics were shown for different lots and strengths of the drug product. Overall, area under the plasma concentration (activity) curve from administration to infinity (AUC) and t1(/2) tended to increase with increasing age, with lower AUC and shorter t(1/2) being seen in children than in adolescents and adults. The PK profiles of turoctocog alfa and other commercially available plasma-derived FVIII and rFVIII products were similar in all age groups. The PK characteristics of turoctocog alfa have been thoroughly studied, and shown to be consistent over time, reproducible between different lots and strengths of drug product, and similar to those observed for other FVIII products. © 2014 International Society on Thrombosis and Haemostasis.

  16. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  17. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    Science.gov (United States)

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

    Science.gov (United States)

    Jaikua, Wipaphorn; Kueakhai, Pornanan; Chaithirayanon, Kulathida; Tanomrat, Rataya; Wongwairot, Sirima; Riengrojpitak, Suda; Sobhon, Prasert; Changklungmoa, Narin

    2016-10-01

    Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50μg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Cytosolic adenylate changes during exercise in prawn muscle

    International Nuclear Information System (INIS)

    Thebault, M.T.; Raffin, J.P.; Pichon, R.

    1994-01-01

    31 P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

  20. Cloning and characterization of human liver cytosolic beta-glycosidase

    NARCIS (Netherlands)

    De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

    2001-01-01

    Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

  1. The Arabidopsis cytosolic proteome

    DEFF Research Database (Denmark)

    Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

    2014-01-01

    compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

  2. NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Jinmin Gao

    2015-03-01

    Full Text Available The formation of DNA double-strand breaks (DSBs must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

  3. NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

    Science.gov (United States)

    Gao, Jinmin; Kim, Hyun-Min; Elia, Andrew E; Elledge, Stephen J; Colaiácovo, Monica P

    2015-03-01

    The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

  4. A cytosolic juxtamembrane interface modulates plexin A3 oligomerization and signal transduction.

    Directory of Open Access Journals (Sweden)

    Rachael Barton

    Full Text Available Plexins (plxns are transmembrane (TM receptors involved in the guidance of vascular, lymphatic vessel, and neuron growth as well as cancer metastasis. Plxn signaling results in cytosolic GTPase-activating protein activity, and previous research implicates dimerization as important for activation of plxn signaling. Purified, soluble plxn extracellular and cytosolic domains exhibit only weak homomeric interactions, suggesting a role for the plxn TM and juxtamembrane regions in homooligomerization. In this study, we consider a heptad repeat in the Danio rerio PlxnA3 cytosolic juxtamembrane domain (JM for its ability to influence PlxnA3 homooligomerization in TM-domain containing constructs. Site-directed mutagenesis in conjunction with the AraTM assay and bioluminescent energy transfer (BRET² suggest an interface involving a JM heptad repeat, in particular residue M1281, regulates PlxnA3 homomeric interactions when examined in constructs containing an ectodomain, TM and JM domain. In the presence of a neuropilin-2a co-receptor and semaphorin 3F ligand, disruption to PlxnA3 homodimerization caused by an M1281F mutation is eliminated, suggesting destabilization of the PlxnA3 homodimer in the JM is not sufficient to disrupt co-receptor complex formation. In contrast, enhanced homodimerization of PlxnA3 caused by mutation M1281L remains even in the presence of ligand semaphorin 3F and co-receptor neuropilin-2a. Consistent with this pattern of PlxnA3 dimerization in the presence of ligand and co-receptor, destabilizing mutations to PlxnA3 homodimerization (M1281F are able to rescue motor patterning defects in sidetracked zebrafish embryos, whereas mutations that enhance PlxnA3 homodimerization (M1281L are not. Collectively, our results indicate the JM heptad repeat, in particular residue M1281, forms a switchable interface that modulates both PlxnA3 homomeric interactions and signal transduction.

  5. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  6. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Sasnauskas Kęstutis

    2011-05-01

    Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

  7. Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination

    International Nuclear Information System (INIS)

    Ohara, Maki; Funyu, Yumi; Ebara, Shunsuke

    2014-01-01

    Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy. (author)

  8. Spectrum Recombination.

    Science.gov (United States)

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  9. Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

    Science.gov (United States)

    Pavlik, Benjamin J; Hruska, Elizabeth J; Van Cott, Kevin E; Blum, Paul H

    2016-03-30

    Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

  10. Chromosomal instability drives metastasis through a cytosolic DNA response.

    Science.gov (United States)

    Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C

    2018-01-25

    Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

  11. The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Lykke, Anne; Rasmussen, Lene Juel

    2010-01-01

    Several enzymes of the metabolic pathways responsible for metabolism of cytosolic ribonucleotides and deoxyribonucleotides are located in mitochondria. Studies described in this paper suggest dysfunction of the mitochondria to affect these metabolic pathways and limit the available levels...

  12. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

    Science.gov (United States)

    VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

    2015-01-01

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

  13. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    Science.gov (United States)

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. DMPD: Cytosolic DNA recognition for triggering innate immune responses. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18280611 Cytosolic DNA recognition for triggering innate immune responses. Takaoka ...A, Taniguchi T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):847-57. Epub 2007 Dec 31. (.png) (.svg) (.html) (.csml) Show Cytosol...ic DNA recognition for triggering innate immune responses. PubmedID 18280611 Title Cytosolic D

  15. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  16. Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.

    Science.gov (United States)

    Camillo, Luciana Rodrigues; Filadelfo, Ciro Ribeiro; Monzani, Paulo Sérgio; Corrêa, Ronan Xavier; Gramacho, Karina Peres; Micheli, Fabienne; Pirovani, Carlos Priminho

    2013-12-01

    The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao-M. perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  17. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

  18. Domains and domain loss

    DEFF Research Database (Denmark)

    Haberland, Hartmut

    2005-01-01

    politicians and in the media, especially in the discussion whether some languages undergo ‘domain loss’ vis-à-vis powerful international languages like English. An objection that has been raised here is that domains, as originally conceived, are parameters of language choice and not properties of languages...

  19. Effect of diphtheria toxin T-domain on endosomal pH

    Directory of Open Access Journals (Sweden)

    A. J. Labyntsev

    2015-08-01

    Full Text Available A key step in the mode of cytotoxic action of diphtheria toxin (DT is the transfer of its catalytic domain (Cd from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td, but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

  20. Recombinant Cyclophilins Lack Nuclease Activity

    OpenAIRE

    Manteca, Angel; Sanchez, Jesus

    2004-01-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

  1. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

    Science.gov (United States)

    Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

    2017-08-01

    One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

  2. Mechanistic logic underlying the axonal transport of cytosolic proteins

    Science.gov (United States)

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  3. Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

    Science.gov (United States)

    Schröder, Katrin; Weissmann, Norbert; Brandes, Ralf P

    2017-08-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Chondroitin sulphate A (CSA)-binding of single recombinant Duffy-binding-like domains is not restricted to Plasmodium falciparum Erythrocyte Membrane Protein 1 expressed by CSA-binding parasites

    DEFF Research Database (Denmark)

    Resende, Mafalda; Ditlev, Sisse B; Nielsen, Morten A

    2009-01-01

    Individuals living in areas with high Plasmodium falciparum transmission acquire immunity to malaria over time and adults have a markedly reduced risk of contracting severe disease. However, pregnant women constitute an important exception. Pregnancy-associated malaria is a major cause of mother....... In this study, we confirm the CSA-binding of these DBL domains, however, the analysis of a number of DBL domains of a non-VAR2CSA origin shows that CSA-binding is not exclusively restricted to VAR2CSA DBL domains. Furthermore, we show that the VAR2CSA DBL domains as well as other DBL domains also bind heparan...

  5. The extent and importance of intragenic recombination

    Directory of Open Access Journals (Sweden)

    de Silva Eric

    2004-11-01

    Full Text Available Abstract We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

  6. Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

    DEFF Research Database (Denmark)

    Liu, Xiangdong; Martens, Helle; Schulz, Alexander

    Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

  7. The chaperone BAG6 captures dislocated glycoproteins in the cytosol.

    Directory of Open Access Journals (Sweden)

    Jasper H L Claessen

    Full Text Available Secretory and membrane (glycoproteins are subject to quality control in the endoplasmic reticulum (ER to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle.

  8. A new view of the bacterial cytosol environment.

    Directory of Open Access Journals (Sweden)

    Benjamin P Cossins

    2011-06-01

    Full Text Available The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg(2+ ions were prominent in NIMS and almost absent free in solution. Κ(+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution.

  9. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  10. Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

    Science.gov (United States)

    Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

    2004-07-01

    The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

  11. Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

    Directory of Open Access Journals (Sweden)

    Yu-Fu Hung

    2015-07-01

    Full Text Available Dengue virus (DENV is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A is a vital component of the viral replication complex (RC and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48 is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48 to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1–48 to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.

  12. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  13. Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme

    Directory of Open Access Journals (Sweden)

    Karla Ramirez-Estrada

    2017-06-01

    Full Text Available Sterol glycosyltransferases (SGTs catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic

  14. Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

    Science.gov (United States)

    Aguayo, María Francisca; Ampuero, Diego; Mandujano, Patricio; Parada, Roberto; Muñoz, Rodrigo; Gallart, Marta; Altabella, Teresa; Cabrera, Ricardo; Stange, Claudia; Handford, Michael

    2013-05-01

    Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. The YARHG domain: an extracellular domain in search of a function.

    Directory of Open Access Journals (Sweden)

    Penny Coggill

    Full Text Available We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times.

  16. Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

    Science.gov (United States)

    Iurescia, Sandra; Fioretti, Daniela; Rinaldi, Monica

    2018-01-01

    The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8 + T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways

  17. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    Science.gov (United States)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  18. Protein abundance profiling of the Escherichia coli cytosol

    DEFF Research Database (Denmark)

    Ishihama, Y.; Schmidt, T.; Rappsilber, J.

    2008-01-01

    sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI...... approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal...

  19. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  20. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  1. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Vemuri, G. N.; Bao, X. M.

    2009-01-01

    of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase...

  2. Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain

    DEFF Research Database (Denmark)

    Pedersen, Henrik; Carlsen, Morten; Nielsen, Jens Bredal

    1999-01-01

    Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized,vith respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition...

  3. Domain analysis

    DEFF Research Database (Denmark)

    Hjørland, Birger

    2017-01-01

    The domain-analytic approach to knowledge organization (KO) (and to the broader field of library and information science, LIS) is outlined. The article reviews the discussions and proposals on the definition of domains, and provides an example of a domain-analytic study in the field of art studies....... Varieties of domain analysis as well as criticism and controversies are presented and discussed....

  4. Cellulose binding domain fusion proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  5. Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

    Science.gov (United States)

    Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

    2009-04-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

  6. Recombinant Breast Cancer Vaccines

    National Research Council Canada - National Science Library

    Pilon, Shari

    1999-01-01

    .... To generate cytosolic proteins, (cytE2, cytE2A), the ER signal sequence was deleted. Vaccination of BALB/c mice with DNA encoding transmembrane E2 or E2A induced anti-ErbB-2 antibodies and anti-tumor immunity, with E2 being more potent than E2A...

  7. Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

    Directory of Open Access Journals (Sweden)

    Joseph A Ross

    2011-07-01

    Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

  8. Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

    International Nuclear Information System (INIS)

    Petrovic, Uros; Sribar, Jernej; Paris, Alenka; Rupnik, Marjan; Krzan, Mojca; Vardjan, Nina; Gubensek, Franc; Zorec, Robert; Krizaj, Igor

    2004-01-01

    Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A 2 acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment

  9. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  10. Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

    Directory of Open Access Journals (Sweden)

    Luis Andre Mariuba

    2008-09-01

    Full Text Available Rhoptry-associated protein 2 (RAP2 is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2 was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

  11. A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

    OpenAIRE

    Jafri, M S; Vajda, S; Pasik, P; Gillo, B

    1992-01-01

    Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering ...

  12. Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

    Directory of Open Access Journals (Sweden)

    Tobias Haase

    Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

  13. The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

    Science.gov (United States)

    Camire, Eric J; Grossman, John D; Thole, Grace J; Fleischman, Nicholas M; Perlstein, Deborah L

    2015-09-25

    Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Recombination of cluster ions

    Science.gov (United States)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  15. Concrete domains

    OpenAIRE

    Kahn, G.; Plotkin, G.D.

    1993-01-01

    This paper introduces the theory of a particular kind of computation domains called concrete domains. The purpose of this theory is to find a satisfactory framework for the notions of coroutine computation and sequentiality of evaluation.

  16. Structural characterization of coatomer in its cytosolic state

    Directory of Open Access Journals (Sweden)

    Shengliu Wang

    2016-07-01

    Full Text Available Abstract Studies on coat protein I (COPI have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

  17. Protein domain organisation: adding order

    Directory of Open Access Journals (Sweden)

    Kummerfeld Sarah K

    2009-01-01

    Full Text Available Abstract Background Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. Results We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Conclusion Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected

  18. Protein domain organisation: adding order.

    Science.gov (United States)

    Kummerfeld, Sarah K; Teichmann, Sarah A

    2009-01-29

    Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and

  19. Domain Engineering

    Science.gov (United States)

    Bjørner, Dines

    Before software can be designed we must know its requirements. Before requirements can be expressed we must understand the domain. So it follows, from our dogma, that we must first establish precise descriptions of domains; then, from such descriptions, “derive” at least domain and interface requirements; and from those and machine requirements design the software, or, more generally, the computing systems.

  20. A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

    Science.gov (United States)

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

    2006-01-01

    Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

  1. Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Marcos Antonio de, E-mail: scaff@lnls.br; Genu, Victor; Discola, Karen Fulan; Alves, Simone Vidigal; Netto, Luis Eduardo Soares [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-900 São Paulo-SP (Brazil); Guimarães, Beatriz Gomes, E-mail: scaff@lnls.br [Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, 13084-971 Campinas-SP (Brazil); Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-900 São Paulo-SP (Brazil)

    2007-08-01

    A recombinant mutant (C47S) of cytosolic thioredoxin peroxidase 1 from S. cerevisiae was expressed, purified and crystallized by the hanging-drop vapour-diffusion method from protein previously treated with 1,4-dithiothreitol. The crystals belong to the monoclinic space group C2 and diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His{sub 6}-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.

  2. Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

    Science.gov (United States)

    Strižak, Zeljka; Ivanković, Dušica; Pröfrock, Daniel; Helmholz, Heike; Cindrić, Ana-Marija; Erk, Marijana; Prange, Andreas

    2014-02-01

    Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules. © 2013.

  3. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rimsa, Vadim; Eadsforth, Thomas C. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Joosten, Robbie P. [Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam (Netherlands); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  4. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    International Nuclear Information System (INIS)

    Rimsa, Vadim; Eadsforth, Thomas C.; Joosten, Robbie P.; Hunter, William N.

    2014-01-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn 2+ -containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn 2+ , where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate

  5. Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

    DEFF Research Database (Denmark)

    Satrústegui, Jorgina; Bak, Lasse K

    2015-01-01

    that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium...

  6. Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

    OpenAIRE

    Tomokazu, Konishi; Takahiro, Kamoi; Ryuichi, Matsuno; Yukiko, Sasaki; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural College of Agriculture; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Plant Molecular Biology, School of Agricultural Sciences, Nagoya University

    1996-01-01

    Levels of subunits of two acetyl-coenzyme A carboxylases were high in small leaves of Pisum sativum, decreased with growth, and remained constant in fully expanded leaves. Irradiation of fully expanded leaves induced the cytosolic isozyme only. This result suggests a key role for the cytosolic enzyme in protection against UV-B.

  7. Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization.

    Science.gov (United States)

    Veeranki, Sudhakar; Choubey, Divaker

    2012-01-01

    The interferon (IFN)-inducible p200-protein family includes structurally related murine (for example, p202a, p202b, p204, and Aim2) and human (for example, AIM2 and IFI16) proteins. All proteins in the family share a partially conserved repeat of 200-amino acid residues (also called HIN-200 domain) in the C-terminus. Additionally, most proteins (except the p202a and p202b proteins) also share a protein-protein interaction pyrin domain (PYD) in the N-terminus. The HIN-200 domain contains two consecutive oligosaccharide/oligonucleotide binding folds (OB-folds) to bind double stranded DNA (dsDNA). The PYD domain in proteins allows interactions with the family members and an adaptor protein ASC. Upon sensing cytosolic dsDNA, Aim2, p204, and AIM2 proteins recruit ASC protein to form an inflammasome, resulting in increased production of proinflammatory cytokines. However, IFI16 protein can sense cytosolic as well as nuclear dsDNA. Interestingly, the IFI16 protein contains a nuclear localization signal (NLS). Accordingly, the initial studies had indicated that the endogenous IFI16 protein is detected in the nucleus and within the nucleus in the nucleolus. However, several recent reports suggest that subcellular localization of IFI16 protein in nuclear versus cytoplasmic (or both) compartment depends on cell type. Given that the IFI16 protein can sense cytosolic as well as nuclear dsDNA and can initiate different innate immune responses (production of IFN-β versus proinflammatory cytokines), here we evaluate the experimental evidence for the regulation of subcellular localization of IFI16 protein in various cell types. We conclude that further studies are needed to understand the molecular mechanisms that regulate the subcellular localization of IFI16 protein. Published by Elsevier Ltd.

  8. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can

  9. Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS.

    Science.gov (United States)

    Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

    2016-01-01

    Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

  10. Cytosolic lipolysis and lipophagy: two sides of the same coin.

    Science.gov (United States)

    Zechner, Rudolf; Madeo, Frank; Kratky, Dagmar

    2017-11-01

    Fatty acids are the most efficient substrates for energy production in vertebrates and are essential components of the lipids that form biological membranes. Synthesis of triacylglycerols from non-esterified free fatty acids (FFAs) combined with triacylglycerol storage represents a highly efficient strategy to stockpile FFAs in cells and prevent FFA-induced lipotoxicity. Although essentially all vertebrate cells have some capacity to store and utilize triacylglycerols, white adipose tissue is by far the largest triacylglycerol depot and is uniquely able to supply FFAs to other tissues. The release of FFAs from triacylglycerols requires their enzymatic hydrolysis by a process called lipolysis. Recent discoveries thoroughly altered and extended our understanding of lipolysis. This Review discusses how cytosolic 'neutral' lipolysis and lipophagy, which utilizes 'acid' lipolysis in lysosomes, degrade cellular triacylglycerols as well as how these pathways communicate, how they affect lipid metabolism and energy homeostasis and how their dysfunction affects the pathogenesis of metabolic diseases. Answers to these questions will likely uncover novel strategies for the treatment of prevalent metabolic diseases.

  11. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

    Science.gov (United States)

    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  12. Cytosolic access of intracellular bacterial pathogens: the Shigella paradigm

    Directory of Open Access Journals (Sweden)

    Nora eMellouk

    2016-04-01

    Full Text Available Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  13. Cytosolic sensing of immuno-stimulatory DNA, the enemy within.

    Science.gov (United States)

    Dhanwani, Rekha; Takahashi, Mariko; Sharma, Sonia

    2018-02-01

    In the cytoplasm, DNA is sensed as a universal danger signal by the innate immune system. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor/enzyme that catalyzes formation of 2'-5'-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates the Stimulator of Interferon Genes (STING), resulting in recruitment of Tank Binding Kinase 1 (TBK1), activation of the transcription factor Interferon Regulatory Factor 3 (IRF3), and trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I). Activation of the pro-inflammatory cGAS-STING-IRF3 response is triggered by direct recognition of the DNA genomes of bacteria and viruses, but also during RNA virus infection, neoplastic transformation, tumor immunotherapy and systemic auto-inflammatory diseases. In these circumstances, the source of immuno-stimulatory DNA has often represented a fundamental yet poorly understood aspect of the response. This review focuses on recent findings related to cGAS activation by an array of self-derived DNA substrates, including endogenous retroviral elements, mitochondrial DNA (mtDNA) and micronuclei generated as a result of genotoxic stress and DNA damage. These findings emphasize the role of the cGAS axis as a cell-intrinsic innate immune response to a wide variety of genomic insults. Copyright © 2017. Published by Elsevier Ltd.

  14. Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Yu J. Shen

    2015-04-01

    Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  15. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Hadron correlations from recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    Quark recombination is a successful model to describe the hadronization of a deconfined quark gluon plasma. Jet-like dihadron correlations measured at RHIC provide a challenge for this picture. We discuss how correlations between hadrons can arise from correlations between partons before hadronization. An enhancement of correlations through the recombination process, similar to the enhancement of elliptic flow is found. Hot spots from completely or partially quenched jets are a likely source of such parton correlations.

  17. DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

  18. Cytosolic PrP Can Participate in Prion-Mediated Toxicity

    Science.gov (United States)

    Thackray, Alana M.; Zhang, Chang; Arndt, Tina

    2014-01-01

    ABSTRACT Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCE During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and

  19. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    International Nuclear Information System (INIS)

    Golubnitchaya-Labudova, O.; Hoefer, M.; Portele, A.; Vacata, V.; Rink, H.; Lubec, G.

    1997-01-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer the question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the inserts of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9. (author)

  20. Cytosolic Calcium Coordinates Mitochondrial Energy Metabolism with Presynaptic Activity

    Science.gov (United States)

    Chouhan, Amit K.; Ivannikov, Maxim V.; Lu, Zhongmin; Sugimori, Mutsuyuki; Llinas, Rodolfo R.; Macleod, Gregory T.

    2012-01-01

    Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations which blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+, and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329±11nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13nM). In summary, we show that when MNs fire at endogenous rates [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs. PMID:22279208

  1. PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration▿ †

    OpenAIRE

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-01-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin inv...

  2. Vitamin A effects on UMR 106 osteosarcoma cells are not mediated by specific cytosolic receptors.

    OpenAIRE

    Oreffo, R O; Francis, J A; Triffitt, J T

    1985-01-01

    Retinol and retinoic acid at 20 microM altered cell morphology and inhibited cell proliferation of UMR 106 osteosarcoma cells in culture. No specific cytosolic binding proteins for retinol could be detected.

  3. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  4. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

  5. A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei.

    OpenAIRE

    Guarnieri, C; Flamigni, F; Caldarera, C M

    1980-01-01

    An alpha-tocopherol-binding macromolecule was isolated from the heart cytosol of rabbits fed for 1 month with an alpha-tocopherol-deficient diet. The amount of [3H]-tocopherol bound to nuclear chromatin was increased when the alpha-tocopherol-deficient heart nuclei were incubated in the presence of [3H]tocopherol-cytosol complex. In this condition, large amounts of [3H]tocopherol were associated with a subnuclear fraction that contained non-histone acidic proteins.

  6. Recombinational repair: workshop summary

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1983-01-01

    Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

  7. MSD Recombination Method in Statistical Machine Translation

    Science.gov (United States)

    Gros, Jerneja Žganec

    2008-11-01

    Freely available tools and language resources were used to build the VoiceTRAN statistical machine translation (SMT) system. Various configuration variations of the system are presented and evaluated. The VoiceTRAN SMT system outperformed the baseline conventional rule-based MT system in all English-Slovenian in-domain test setups. To further increase the generalization capability of the translation model for lower-coverage out-of-domain test sentences, an "MSD-recombination" approach was proposed. This approach not only allows a better exploitation of conventional translation models, but also performs well in the more demanding translation direction; that is, into a highly inflectional language. Using this approach in the out-of-domain setup of the English-Slovenian JRC-ACQUIS task, we have achieved significant improvements in translation quality.

  8. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  9. Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

    Science.gov (United States)

    Santos, Clelton A; Saraiva, Antonio M; Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Favaro, Marianna T P; Horta, Maria A C; Santiago, André S; Mendes, Juliano S; Souza, Alessandra A; Souza, Anete P

    2013-01-01

    The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Identification and characterization of two novel cytosolic sulfotransferases, SULT1 ST7 and SULT1 ST8, from zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Liu, T.-A. [Department of Pharmacology, College of Pharmacy, University of Toledo, Toledo, OH 43606 (United States); Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan (China); Bhuiyan, Shakhawat [Division of Arts and Sciences, Jarvis Christian College, Hawkins, TX 75765 (United States); Snow, Rhodora [School of Mathematics and Science, J. Sargeant Reynolds Community College, Richmond, VA 23285 (United States); Yasuda, Shin; Yasuda, Tomoko [Department of Pharmacology, College of Pharmacy, University of Toledo, Toledo, OH 43606 (United States); Yang, Y.-S. [Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan (China); Williams, Frederick E.; Liu, M.-Y.; Suiko, Masahito [Department of Pharmacology, College of Pharmacy, University of Toledo, Toledo, OH 43606 (United States); Carter, Glendora [School of Mathematics and Science, J. Sargeant Reynolds Community College, Richmond, VA 23285 (United States); Liu, M.-C. [Department of Pharmacology, College of Pharmacy, University of Toledo, Toledo, OH 43606 (United States)], E-mail: ming.liu@utoledo.edu

    2008-08-29

    Cytosolic sulfotransferases (SULTs) constitute a family of Phase II detoxification enzymes that are involved in the protection against potentially harmful xenobiotics as well as the regulation and homeostasis of endogenous compounds. Compared with humans and rodents, the zebrafish serves as an excellent model for studying the role of SULTs in the detoxification of environmental pollutants including environmental estrogens. By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative SULTs were identified. Sequence analysis indicated that these two putative zebrafish SULTs belong to the SULT1 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT1 ST7 and SULT1 ST8, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) cells. Purified GST-fusion protein form of SULT1 ST7 and SULT1 ST8 exhibited strong sulfating activities toward environmental estrogens, particularly hydroxylated polychlorinated biphenyls (PCBs), among various endogenous and xenobiotic compounds tested as substrates. pH-dependence experiments showed that SULT1 ST7 and SULT1 ST8 displayed pH optima at 6.5 and 8.0, respectively. Kinetic parameters of the two enzymes in catalyzing the sulfation of catechin and chlorogenic acid as well as 3-chloro-4-biphenylol were determined. Developmental expression experiments revealed distinct patterns of expression of SULT1 ST7 and SULT1 ST8 during embryonic development and throughout the larval stage onto maturity.

  11. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Parton recombination model

    International Nuclear Information System (INIS)

    Hwa, R.C.

    1978-08-01

    Low P/sub T/ meson production in hadronic collisions is described in the framework of the parton model. The recombination of quark and antiquark is suggested as the dominant mechanism in the large x region. Phenomenological evidences for the mechanism are given. The application to meson initiated reactions yields the quark distribution in mesons. 21 references

  13. Domain crossing

    DEFF Research Database (Denmark)

    Schraefel, M. C.; Rouncefield, Mark; Kellogg, Wendy

    2012-01-01

    In CSCW, how much do we need to know about another domain/culture before we observe, intersect and intervene with designs. What optimally would that other culture need to know about us? Is this a “how long is a piece of string” question, or an inquiry where we can consider a variety of contexts a...

  14. Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

    International Nuclear Information System (INIS)

    Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

    1986-01-01

    Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

  15. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

    Directory of Open Access Journals (Sweden)

    Helena H. Chowdhury

    2014-10-01

    Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  16. Nucleic acids encoding a cellulose binding domain

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  17. Recombinant expression and purification of L2 domain of human ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... in cell multiplication processes and seem as good targets for interventional therapies. EGFR is a receptor tyrosine kinase that expresses in a significantly higher level in several types of cancer cells and its activation results in cell proliferation, differentiation, cell adhesion, migration and angiogenesis.

  18. A Case Study of a Multiobjective Elitist Recombinative Genetic Algorithm with Coevolutionary Sharing

    NARCIS (Netherlands)

    Neef, R.M.; Thierens, D.; Arciszewski, H.F.R.

    1999-01-01

    We present a multiobjective genetic algorithm that incorporates various genetic algorithm techniques that have been proven to be efficient and robust in their problem domain. More specifically, we integrate rank based selection, adaptive niching through coevolutionary sharing, elitist recombination,

  19. A case study of a multiobjective recombinative genetic algorithm with coevolutionary sharing

    NARCIS (Netherlands)

    Neef, R.M.; Thierens, D.; Arciszewski, H.F.R.

    1999-01-01

    We present a multiobjective genetic algorithm that incorporates various genetic algorithm techniques that have been proven to be efficient and robust in their problem domain. More specifically, we integrate rank based selection, adaptive niching through coevolutionary sharing, elitist recombination,

  20. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

    Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

  1. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

    KAUST Repository

    Houben, Diane; Demangel, Caroline; Van Ingen, Jakko; Perez, Jorge; Baldeó n, Lucy R.; Abdallah, Abdallah; Caleechurn, Laxmee; Bottai, Daria; Van Zon, Maaike; De Punder, Karin; Van Der Laan, Tridia; Kant, Arie; Bossers-De Vries, Ruth; Willemsen, Peter Th J; Bitter, Wilbert M.; Van Soolingen, Dick; Brosch, Roland; Van Der Wel, Nicole N.; Peters, Peter J.

    2012-01-01

    Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

  2. Circadian Rhythm of Hepatic Cytosolic and Nuclear Estrogen and Androgen Receptors

    Science.gov (United States)

    FRANCAVILLA, ANTONIO; EAGON, PATRICIA K.; DiLEO, ALFREDO; VAN THIEL, DAVID H.; PANELLA, CARMINE; POLIMENO, LORENZO; AMORUSO, CINZIA; INGROSSO, MARCELLO; AQUILINO, A. MARIA; STARZL, THOMAS E.

    2010-01-01

    Mammalian liver is a sex steroid-responsive tissue. The effects of these hormones presumably are mediated by hepatic estrogen receptors (ER) and androgen receptors (AR). Serum levels of sex hormones display circadian rhythms. Further, estrogens and androgens are commonly administered; administration of these agents is associated frequently with liver disease. Therefore, we investigated whether the cytosolic and nuclear sex steroid receptors also display a similar circadian rhythm, and whether variations occurred in the distribution of receptors between cytosolic and nuclear compartments. Animals were killed every 4 h from midnight till the following midnight; cytosolic and nuclear levels of both ER and AR were measured. Cytosolic ER reached a maximum level at 4 AM, and a minimum at 8 PM and midnight of both days. Nuclear ER was highest at 8 AM and lowest at 4 PM and 8 PM, a pattern which parallels variations in serum estradiol levels. Cytosolic AR was highest at 8 PM and lowest at midnight and 4 AM. Nuclear AR was highest at 4 AM and lowest at 4 PM and 8 PM. The highest level of nuclear AR does not correspond to the maximum serum testosterone level, which occurred at 4 PM. The total hepatic content of both ER and AR was not constant over the 24-h period, but varied considerably with time of day. These studies suggest that both ER and AR show a distinct circadian rhythm in subcellular compartmentalization, and that total hepatic content of ER and AR varies significantly during a 24-h period. PMID:3710067

  3. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

    KAUST Repository

    Houben, Diane

    2012-05-08

    Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

  4. Trusted Domain

    DEFF Research Database (Denmark)

    Hjorth, Theis Solberg; Torbensen, Rune

    2012-01-01

    remote access via IP-based devices such as smartphones. The Trusted Domain platform fits existing legacy technologies by managing their interoperability and access controls, and it seeks to avoid the security issues of relying on third-party servers outside the home. It is a distributed system...... of wireless standards, limited resources of embedded systems, etc. Taking these challenges into account, we present a Trusted Domain home automation platform, which dynamically and securely connects heterogeneous networks of Short-Range Wireless devices via simple non-expert user. interactions, and allows......In the digital age of home automation and with the proliferation of mobile Internet access, the intelligent home and its devices should be accessible at any time from anywhere. There are many challenges such as security, privacy, ease of configuration, incompatible legacy devices, a wealth...

  5. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  6. Site directed recombination

    Science.gov (United States)

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  7. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  8. Recombination epoch revisited

    International Nuclear Information System (INIS)

    Krolik, J.H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons. 18 references

  9. Dielectronic recombination theory

    International Nuclear Information System (INIS)

    LaGattuta, K.J.

    1991-01-01

    A theory now in wide use for the calculation of dielectronic recombination cross sections (σ DR ) and rate coefficients (α DR ) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of σ DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of α DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of σ DR . While the measurements of σ DR for δn ≠ 0 excitations have tended to agree very well with calculations, the case of δn = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

  10. The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

    Science.gov (United States)

    Yan, Nan; Regalado-Magdos, Ashton D.; Stiggelbout, Bart; Lee-Kirsch, Min Ae; Lieberman, Judy

    2010-01-01

    Viral infection triggers innate immune sensors to produce type I interferons (IFN). However, HIV infection of T cells and macrophages does not trip these alarms. How HIV avoids activating nucleic acid sensors is unknown. The cytosolic exonuclease TREX1 suppressed IFN triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA interference, cytosolic HIV DNA accumulated, and HIV infection induced type I IFN that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate IFN expression via a TBK1, STING and IRF3 dependent pathway. HIV-stimulated IFN production in cells deficient in TREX1 did not involve known nucleic acid sensors. PMID:20871604

  11. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    Science.gov (United States)

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  12. Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

    Science.gov (United States)

    Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

    1999-04-01

    4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

  13. Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

    2009-01-01

    the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

  14. Functional, Responsive Materials Assembled from Recombinant Oleosin

    Science.gov (United States)

    Hammer, Daniel

    Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

  15. Genetic recombination is directed away from functional genomic elements in mice.

    Science.gov (United States)

    Brick, Kevin; Smagulova, Fatima; Khil, Pavel; Camerini-Otero, R Daniel; Petukhova, Galina V

    2012-05-13

    Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

  16. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    Science.gov (United States)

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver CytosolShan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas S-adenosyl-L-methionine (AdoMet): ar...

  17. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

    Science.gov (United States)

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-05-27

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

  18. Effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice

    International Nuclear Information System (INIS)

    Meng Qingyong; Chen Shali; Liu Shuzheng

    2003-01-01

    Objective: To the effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice. Methods: The expression of proteins was analyzed by gel filtration with Sephadex G-100 and HPLC based on separation of proteins on thymocyte cytosol and nuclei after whole-body irradiation with 75 mGy X-rays and sham-irradiation, and their biological activity was examined by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes. Results: HPLC analysis showed that there was a marked increase in expression of 61.4 kD protein in the extract of thymocyte cytosol and 30.4 kD protein in the extract of thymocyte nuclei in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocyte cytosol and nuclei of the irradiated mice showed both stimulating effect on normal T cell proliferation and protective effect on chromosome damage induced by high dose radiation. Conclusion: These findings might have implications in study of mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation

  19. Nanoparticles for cytosolic delivery of important biomolecular drugs such as DNA, RNA, peptides, and proteins

    Czech Academy of Sciences Publication Activity Database

    Sedlák, M.; Koňák, Čestmír; Dybal, Jiří

    2010-01-01

    Roč. 1, č. 2010 (2010), s. 87-90 ISSN 2210-2892 Institutional research plan: CEZ:AV0Z40500505 Keywords : cytosolic delivery * nanoparticle carriers * poly(ethylacrylic acid) Subject RIV: CD - Macromolecular Chemistry http://benthamopen.com/ABSTRACT/TOPROCJ-1-87

  20. Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-27

    Dec 27, 2010 ... The neutrophil cytosol factor 1 (NCF1) gene consists of 11 exons and is found in two forms; one is wild ... granulomatous disease, multiple sclerosis, arthritis and parasitic infection. ... TCR, T cell receptor; AhR, aryl hydrocarbon receptor; RA, .... During malaria, ROS production can contribute to both.

  1. The role of a cytosolic superoxide dismutase in barley-pathogen interactions

    KAUST Repository

    Lightfoot, Damien; Mcgrann, Graham R. D.; Able, Amanda J.

    2016-01-01

    susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2-HO2 contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi

  2. Recombinant Innovation and Endogenous Transitions

    OpenAIRE

    Koen Frenken; Luis R. Izquierdo; Paolo Zeppini

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create “short-cuts” which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

  3. RPA homologs and ssDNA processing during meiotic recombination.

    Science.gov (United States)

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  4. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  5. Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells

    NARCIS (Netherlands)

    Gankema, H. S.; Groen, A. K.; Wanders, R. J.; Tager, J. M.

    1983-01-01

    1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins,

  6. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  7. On the relict recombination lines

    International Nuclear Information System (INIS)

    Bershtejn, I.N.; Bernshtejn, D.N.; Dubrovich, V.K.

    1977-01-01

    Accurate numerical calculation of intensities and profiles of hydrogen recombination lines of cosmological origin is made. Relie radiation distortions stipulated by recombination quantum release at the irrevocable recombination are investigated. Mean number calculation is given for guantums educing for one irrevocably-lost electron. The account is taken of the educed quantums interraction with matter. The main quantum-matter interrraction mechanisms are considered: electronic blow broadening; free-free, free-bound, bound-bound absorptions Recombination dynamics is investigated depending on hydrogen density and total density of all the matter kinds in the Universe

  8. Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

    Directory of Open Access Journals (Sweden)

    Chiu-Ping Fang

    Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

  9. Species-Specific Monoclonal Antibodies to Escherichia coli-Expressed p36 Cytosolic Protein of Mycoplasma hyopneumoniae

    Science.gov (United States)

    Caron, J.; Sawyer, N.; Moumen, B. Ben Abdel; Bouh, K. Cheikh Saad; Dea, S.

    2000-01-01

    The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an

  10. Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction.

    Directory of Open Access Journals (Sweden)

    Wolfgang Giese

    2018-04-01

    Full Text Available The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus.

  11. Limits to anaerobic energy and cytosolic concentration in the living cell

    Science.gov (United States)

    Paglietti, A.

    2015-11-01

    For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

  12. Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis

    DEFF Research Database (Denmark)

    Kojic, Milorad; Zhou, Qingwen; Lisby, Michael

    2005-01-01

    after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates...

  13. The effects of irradiation on the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver

    International Nuclear Information System (INIS)

    Teshima, Teruki; Mori, Masaki; Honke, Yoshifumi

    1983-01-01

    The effects of irradiation on both the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver were investigated. The liver concentrations of corticosterone and cyclic nucleotides were measured by radioimmunoassay before and after the irradiation of 1,000 rad/l fraction. The glucocorticoid receptor in the liver cytosol was determined by the measurement of the cytosol binding to 3 H-dexamethasone. The cytosol and nuclear corticosterone levels reached a peak 1 day after the irradiation of the rat liver and declined to the control levels after 2 days. The increase in corticosterone levels may be due to the direct stimulation of the right adrenal gland and/ or the stress induced by the irradiation. The binding capacity of the glucocorticoid receptor in rat liver cytosol decreased to the minimum 1 day after the irradiation, and the recovery occurred at 4 days. The Kd value of the glucocorticoid receptor remained unchanged from 1 hour until 4 days but was high at 4 and 7 days. The distinctly increased levels of cyclic GMP in the rat liver were found from 1 hour through 7 days after the irradiation, while cyclic AMP did not change. The inversed relationship between the cytosol glucocorticoid receptor and corticosterone levels in cytosol and the nuclei indicates that the receptor-bound corticosterone in cytosol can be transferred to a nucleus and remain there in the presence of appropriate amounts of corticosterone in cytosol, after which the receptor is released from the nucleus into cytosol. The high Kd values observed 4 -- 7 days after the irradiation may be either due to the direct effect of irradiation or to the replenishment of the receptor with a low affinity. (author)

  14. Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

    Science.gov (United States)

    Arthur, Christopher P; Stowell, Michael H B

    2007-06-01

    Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

  15. Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

    Science.gov (United States)

    Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

    2017-01-01

    Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

  16. .Gov Domains API

    Data.gov (United States)

    General Services Administration — This dataset offers the list of all .gov domains, including state, local, and tribal .gov domains. It does not include .mil domains, or other federal domains outside...

  17. Dissociative recombination of dications

    International Nuclear Information System (INIS)

    Seiersen, K.; Heber, O.; Jensen, M.J.; Safvan, C.P.; Andersen, L. H.

    2003-01-01

    Dissociative recombination (DR) of doubly-charged positive ions has been studied at the heavy ion storage ring ASTRID. Low-energy electrons were scattered on the dication of the N 2 molecule, and the absolute cross section was measured in the energy range of 10 -4 -50 eV. From the measured cross section, a thermal rate coefficient of 5.8x10 -7 cm 3 s -1 at 300 K was extracted. Furthermore, we present new results on the CO 2+ DR rate, and a summary and comparison of measured DR rate coefficients for both the singly and doubly-charged ions of CO, CO 2 , and N 2 is presented

  18. Hadron Correlations and Parton Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: rjfries@comp.tamu.edu

    2007-02-15

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  19. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  20. Auger recombination in sodium iodide

    Science.gov (United States)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  1. Glucose acutely reduces cytosolic and mitochondrial H2O2 in rat pancreatic beta-cells.

    Science.gov (United States)

    Deglasse, Jean-Philippe; Roma, Leticia Prates; Pastor-Flores, Daniel; Gilon, Patrick; Dick, Tobias P; Jonas, Jean-Christophe

    2018-05-14

    Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion by pancreatic β-cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 and HyPer with its pH-control SypHer, to test the acute effects of glucose, monomethylsuccinate, leucine with glutamine, and α-ketoisocaproate, on β-cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5mM glucose in the cytosol and 10mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15µM) did not affect insulin secretion. By contrast, menadione (1-5µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20mM glucose. Subcellular changes in β-cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in β-cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. The glucose-dependent stimulation of insulin secretion occurs independently of a detectable increase in β-cell cytosolic or mitochondrial H2O2 levels.

  2. Purification, crystallization and preliminary crystallographic characterization of the caspase-recruitment domain of human Nod1

    International Nuclear Information System (INIS)

    Srimathi, Thiagarajan; Robbins, Sheila L.; Dubas, Rachel L.; Seo, Jang-Hoon; Park, Young Chul

    2006-01-01

    The caspase-recruitment domain of the cytosolic pathogen receptor Nod1 was crystallized. X-ray diffraction data were collected to 1.9 Å resolution. The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein–protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P3 1 or P3 2 , with unit-cell parameters a = b = 79.1, c = 80.9 Å. Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit

  3. A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.

    Directory of Open Access Journals (Sweden)

    Lydia Dmochewitz

    Full Text Available BACKGROUND: The C3bot1 protein (~23 kDa from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.

  4. Evolution based on domain combinations: the case of glutaredoxins

    Directory of Open Access Journals (Sweden)

    Herrero Enrique

    2009-03-01

    Full Text Available Abstract Background Protein domains represent the basic units in the evolution of proteins. Domain duplication and shuffling by recombination and fusion, followed by divergence are the most common mechanisms in this process. Such domain fusion and recombination events are predicted to occur only once for a given multidomain architecture. However, other scenarios may be relevant in the evolution of specific proteins, such as convergent evolution of multidomain architectures. With this in mind, we study glutaredoxin (GRX domains, because these domains of approximately one hundred amino acids are widespread in archaea, bacteria and eukaryotes and participate in fusion proteins. GRXs are responsible for the reduction of protein disulfides or glutathione-protein mixed disulfides and are involved in cellular redox regulation, although their specific roles and targets are often unclear. Results In this work we analyze the distribution and evolution of GRX proteins in archaea, bacteria and eukaryotes. We study over one thousand GRX proteins, each containing at least one GRX domain, from hundreds of different organisms and trace the origin and evolution of the GRX domain within the tree of life. Conclusion Our results suggest that single domain GRX proteins of the CGFS and CPYC classes have, each, evolved through duplication and divergence from one initial gene that was present in the last common ancestor of all organisms. Remarkably, we identify a case of convergent evolution in domain architecture that involves the GRX domain. Two independent recombination events of a TRX domain to a GRX domain are likely to have occurred, which is an exception to the dominant mechanism of domain architecture evolution.

  5. Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

    Directory of Open Access Journals (Sweden)

    Sachie Tanaka

    2017-05-01

    Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

  6. A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase.

    Science.gov (United States)

    Shang, Pengcheng; Misra, Saurav; Hause, Ben; Fang, Ying

    2017-07-15

    Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3C pro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event. IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we

  7. Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

    Directory of Open Access Journals (Sweden)

    Ambarish Nag

    Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

  8. Automation of metabolic stability studies in microsomes, cytosol and plasma using a 215 Gilson liquid handler.

    Science.gov (United States)

    Linget, J M; du Vignaud, P

    1999-05-01

    A 215 Gilson liquid handler was used to automate enzymatic incubations using microsomes, cytosol and plasma. The design of automated protocols are described. They were based on the use of 96 deep well plates and on HPLC-based methods for assaying the substrate. The assessment of those protocols was made with comparison between manual and automated incubations, reliability and reproducibility of automated incubations in microsomes and cytosol. Examples of the use of those programs in metabolic studies in drug research, i.e. metabolic screening in microsomes and plasma were shown. Even rapid processes (with disappearance half lives as low as 1 min) can be analysed. This work demonstrates how stability studies can be automated to save time, render experiments involving human biological media less hazardous and may be improve inter-laboratory reproducibility.

  9. Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

    Science.gov (United States)

    Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

    2011-01-01

    Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

  10. Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

    Science.gov (United States)

    Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

    2018-06-05

    The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy.

    Science.gov (United States)

    Dreier, Roland Felix; Santos, José Carlos; Broz, Petr

    2018-01-01

    Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

  12. A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

    Directory of Open Access Journals (Sweden)

    Julie Adam

    2013-05-01

    Full Text Available The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH, predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

  13. Well-defined polypeptide-based systems as non-viral vectors for cytosolic delivery

    OpenAIRE

    Niño Pariente, Amaya

    2017-01-01

    A convenient cytosolic drug delivery constitutes a very powerful tool for the treatment and/or prevention of several relevant human diseases. Along with recent advances in therapeutic technologies based on biomacromolecules (e.g. oligonucleotides or proteins), we also require the development of technologies which improve the transport of therapeutic molecules to the cell of choice. This has led to the emergence of a variety of promising methods over the last 20 years. Despite significant prog...

  14. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

    OpenAIRE

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

    2008-01-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expressio...

  15. BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

    Science.gov (United States)

    Kobayashi, Shouhei; Koujin, Takako; Kojidani, Tomoko; Osakada, Hiroko; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-06-02

    Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF(+) DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

  16. Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

    OpenAIRE

    Martos, C; Plana, M; Guasch, M D; Itarte, E

    1985-01-01

    Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partia...

  17. Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

    Directory of Open Access Journals (Sweden)

    Shilpee Srivastava

    Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

  18. GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

    Science.gov (United States)

    Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

    2013-07-01

    GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.

    Science.gov (United States)

    Lee, Joo-Yong; Hwang, Jung Jin; Park, Mi-Ha; Koh, Jae-Young

    2006-01-01

    Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.

  20. Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

    OpenAIRE

    Buonocore, Linda; Blight, Keril J.; Rice, Charles M.; Rose, John K.

    2002-01-01

    We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein...

  1. Generation of truncated recombinant form of tumor necrosis factor ...

    African Journals Online (AJOL)

    Purpose: To produce truncated recombinant form of tumor necrosis factor receptor 1 (TNFR1), cysteine-rich domain 2 (CRD2) and CRD3 regions of the receptor were generated using pET28a and E. coli/BL21. Methods: DNA coding sequence of CRD2 and CRD3 was cloned into pET28a vector and the corresponding ...

  2. A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

    Science.gov (United States)

    Straw, Megan L; Chaplin, Amanda K; Hough, Michael A; Paps, Jordi; Bavro, Vassiliy N; Wilson, Michael T; Vijgenboom, Erik; Worrall, Jonathan A R

    2018-01-24

    Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P 1 -type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

  3. The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

    Science.gov (United States)

    Yang, Bei

    2012-01-01

    Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication. PMID:22031934

  4. TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing.

    Science.gov (United States)

    Seo, Gil Ju; Kim, Charlotte; Shin, Woo-Jin; Sklan, Ella H; Eoh, Hyungjin; Jung, Jae U

    2018-02-09

    Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity.

  5. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

    Science.gov (United States)

    Takahama, Michihiro; Fukuda, Mitsunori; Ohbayashi, Norihiko; Kozaki, Tatsuya; Misawa, Takuma; Okamoto, Toru; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

    2017-09-19

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Michihiro Takahama

    2017-09-01

    Full Text Available Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5, in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING, the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

  7. Cytosolic calcium homeostasis in fungi: Roles of plasma membrane transport and intracellular sequestration of calcium

    International Nuclear Information System (INIS)

    Miller, A.J.; Vogg, G.; Sanders, D.

    1990-01-01

    Cytosolic free calcium ([Ca 2+ ] c ) has been measured in the mycelial fungus Neurospora crassa with Ca 2+ - selective microelectrodes. The mean value of [Ca 2+ ] c is 92 ± 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca 2+ across the plasma membrane to be estimated as about -60 kJmol -1 - a value that cannot be sustained either by a simple Ca 2+ - ATPase, or, in alkaline conditions, by straightforward H + /Ca 2+ exchange with a stoichiometric ratio of + /Ca 2+ . The authors propose that the most likely alternative mechanism of Ca 2+ efflux is ATP-driven H + /Ca 2+ exchange, with a stoichiometric ratio of at least 2 H + /Ca 2+ . The increase in [Ca 2+ ] c in the presence of CN - at pH 8.4 is compared with 45 Ca 2+ influx under the same conditions. The proportion of entering Ca 2+ remaining free in the cytosol is only 8 x 10 -5 , and since the concentration of available chelation sites on Ca 2+ binding proteins is unlikely to exceed 100 μM, a major role for the fungal vacuole in short-term Ca 2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca 2+ homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force for Ca 2+ uptake into fungal vacuoles

  8. AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca(2+) increase.

    Science.gov (United States)

    Francia, Doriana; Chiltz, Annick; Lo Schiavo, Fiorella; Pugin, Alain; Bonfante, Paola; Cardinale, Francesca

    2011-09-01

    The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca(2+) in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca(2+)-channel blocker (La(3+)) showed that in these cells, MAPK activation was dependent on the cytosolic Ca(2+) increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca(2+) transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  9. Chloride concentrations in human hepatic cytosol and mitochondria are a function of age.

    Science.gov (United States)

    Jahn, Stephan C; Rowland-Faux, Laura; Stacpoole, Peter W; James, Margaret O

    2015-04-10

    We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n = 97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2 ± 62.4 mM; range: 24.7-365.7 mM) were strikingly higher than those in mitochondria (4.2 ± 3.8 mM; range 0.9-22.2 mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

    Directory of Open Access Journals (Sweden)

    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  11. Prognostic significance of cytosolic pS2 content in ovarian tumors

    International Nuclear Information System (INIS)

    Raigoso, P.; Allende, T.; Zeidan, N.; Llana, B.; Bernardo, L.; Roiz, C.; Tejuca, S.; Vazquez, J.; Lamelas, M.L.

    2002-01-01

    Aim: pS2 is an estrogen regulated peptide which has been associated with a good prognosis an with a more favorable response to treatment in breast cancer patients. In ovarian tumors, the expression of pS2 was demonstrated at both mRNA and protein levels. In addition, it has been showed significant association of pS2 with mucinous differentiation or well differentiation grade of the tumors. However, it is little know about the prognostic significance of the pS2 content in ovarian carcinomas. The aims of the present work were to analyze the cytosolic pS2 content in benign and malignant ovarian tumors, its relationship with clinico-pathologic parameters, steroid receptor status, and prognostic significance. Material and Methods: We analysed the cytosolic concentrations of pS2 in 91 specimen ovarian tissues by an immunoradiometric assay (ELSA-pS2, CIS, France). The tissues were 8 normal ovaries, 43 benign tumors and 40 malignant ovarian tumors. The same ovarian tissues processed to pS2 were analyzed to Estrogen (ER) and Progesterone (PgR) Receptor status. These steroid receptors were quantified biochemically following commercial ELISA method (ABBOTT Diagnostics, Germany). The relationship between cytosolic content and clinico-pathologic factors was examined by the Mann-Whitney or Kruskall-Wallis test. Correlation between steroid receptors and pS2 content was calculated with the Spearman test. Survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. Differences were considered significant at 5% probability level. Results: pS2 could be detected in 30 cases (32.9%) with values ranged from 0.04 to 89 ng/mg prt. Only one normal ovary showed detectable levels of pS2 and there were not differences in cytosolic content between benign and malignant ovarian tumors. The pS2 levels were only associated to mucinous differentiation in both benign and malignant ovarian tumors (p=0.029 and p=0.015, respectively). Significantly higher

  12. Oxygen-hydrogen recombination system

    International Nuclear Information System (INIS)

    Sato, Shuichiro; Takejima, Masaki.

    1981-01-01

    Purpose: To avoid reduction in the performance of catalyst used for an oxygen-hydrogen recombiner in the off gas processing system of a nuclear reactor. Constitution: A thermometer is provided for the detection of temperature in an oxygen-hydrogen recombiner. A cooling pipe is provided in the recombiner and cooling medium is introduced externally. The cooling medium may be water or air. In accordance with the detection value from the thermometer, ON-OFF control is carried out for a valve to control the flow rate of the cooling medium thereby rendering the temperature in the recombiner to a predetermined value. This can prevent the catalyst from being exposed to high temperature and avoid the reduction in the performance of the catalyst. (Ikeda, J.)

  13. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  14. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

    1997-01-01

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial

  15. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2015-01-01

    as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases

  16. Review of Parton Recombination Models

    International Nuclear Information System (INIS)

    Bass, Steffen A

    2006-01-01

    Parton recombination models have been very successful in explaining data taken at RHIC on hadron spectra and emission patterns in Au+Au collisions at transverse momenta above 2 GeV/c, which have exhibited features which could not be understood in the framework of basic perturbative QCD. In this article I will review the current status on recombination models and outline which future challenges need to be addressed by this class of models

  17. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  18. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-01-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n s , and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z * =1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ε α i <0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  19. Studies of the activity of cytosol on the mixed disulfide bond formed by proteins and radioprotector mercaptoethylguanidine

    Energy Technology Data Exchange (ETDEWEB)

    Horvath, M [National Inst. of Oncology, Budapest (Hungary); Holland, J [Orszagos Onkologiai Intezet, Budapest (Hungary)

    1979-01-01

    The cytoplasm of normal and tumorous rat liver cells contains a heat-resistant compound with reducing ability to break the mixed disulfide bond of albumin-/sup 14/C-mercaptoethylguanidine. The reducing activity of cytosol is destoryed by 1000 krd /sup 60/Co-gamma-ray doses in diluted solution. In vivo supralethal of rats does not affect the activity of cytosol prepared from liver cells.

  20. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    International Nuclear Information System (INIS)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

    2007-01-01

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

  1. Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

    Science.gov (United States)

    Zhang, Xuanbo; Wang, Lihong; Zhou, Anhua; Zhou, Qing; Huang, Xiaohua

    2016-04-01

    The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. The consequences of sequence erosion in the evolution of recombination hotspots.

    Science.gov (United States)

    Tiemann-Boege, Irene; Schwarz, Theresa; Striedner, Yasmin; Heissl, Angelika

    2017-12-19

    Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans -acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'. © 2017 The Authors.

  3. A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

    Science.gov (United States)

    Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A; Portman, Jonathan L; Sauer, John-Demian

    2017-03-21

    Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis , are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role

  4. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

    Science.gov (United States)

    Choi, Kyuha; Reinhard, Carsten; Serra, Heïdi; Ziolkowski, Piotr A; Underwood, Charles J; Zhao, Xiaohui; Hardcastle, Thomas J; Yelina, Nataliya E; Griffin, Catherine; Jackson, Matthew; Mézard, Christine; McVean, Gil; Copenhaver, Gregory P; Henderson, Ian R

    2016-07-01

    Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

  5. Quantifying the mechanisms of domain gain in animal proteins.

    Science.gov (United States)

    Buljan, Marija; Frankish, Adam; Bateman, Alex

    2010-01-01

    Protein domains are protein regions that are shared among different proteins and are frequently functionally and structurally independent from the rest of the protein. Novel domain combinations have a major role in evolutionary innovation. However, the relative contributions of the different molecular mechanisms that underlie domain gains in animals are still unknown. By using animal gene phylogenies we were able to identify a set of high confidence domain gain events and by looking at their coding DNA investigate the causative mechanisms. Here we show that the major mechanism for gains of new domains in metazoan proteins is likely to be gene fusion through joining of exons from adjacent genes, possibly mediated by non-allelic homologous recombination. Retroposition and insertion of exons into ancestral introns through intronic recombination are, in contrast to previous expectations, only minor contributors to domain gains and have accounted for less than 1% and 10% of high confidence domain gain events, respectively. Additionally, exonization of previously non-coding regions appears to be an important mechanism for addition of disordered segments to proteins. We observe that gene duplication has preceded domain gain in at least 80% of the gain events. The interplay of gene duplication and domain gain demonstrates an important mechanism for fast neofunctionalization of genes.

  6. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

    Science.gov (United States)

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

    2009-03-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

  7. The role of a cytosolic superoxide dismutase in barley-pathogen interactions

    KAUST Repository

    Lightfoot, Damien

    2016-03-19

    Reactive oxygen species (ROS), including superoxide (O2-HO2) and hydrogen peroxide (H2O2), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi-biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of O2-HO2 to H2O2, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi-biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up-regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2-HO2 contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi-biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.

  8. Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

    Science.gov (United States)

    Huang, Jian; Zhao, Lei; Yang, Ping; Chen, Zhen; Ruan, Xiong Z; Huang, Ailong; Tang, Ni; Chen, Yaxi

    2017-09-15

    Hepatitis B virus (HBV) is designated a "metabolovirus" due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca 2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases.

    Science.gov (United States)

    Rasool, Mohammed I; Bairam, Ahsan F; Kurogi, Katsuhisa; Liu, Ming-Cheh

    2017-10-01

    Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT. The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays. Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media. SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  10. Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

    Science.gov (United States)

    Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

    2014-09-12

    The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

    Science.gov (United States)

    Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

    2014-01-01

    The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

  12. A Network Approach to Analyzing Highly Recombinant Malaria Parasite Genes

    Science.gov (United States)

    Larremore, Daniel B.; Clauset, Aaron; Buckee, Caroline O.

    2013-01-01

    The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences. PMID:24130474

  13. A network approach to analyzing highly recombinant malaria parasite genes.

    Science.gov (United States)

    Larremore, Daniel B; Clauset, Aaron; Buckee, Caroline O

    2013-01-01

    The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences.

  14. A network approach to analyzing highly recombinant malaria parasite genes.

    Directory of Open Access Journals (Sweden)

    Daniel B Larremore

    Full Text Available The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs, and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences.

  15. From Proteomics to Structural Studies of Cytosolic/Mitochondrial-Type Thioredoxin Systems in Barley Seeds

    DEFF Research Database (Denmark)

    Shahpiri, Azar; Svensson, Birte; Finnie, Christine

    2009-01-01

    Thioredoxins (Trx) are ubiquitous proteins that participate in thiol disulfide reactions via two active site cysteine residues, allowing Trx to reduce disulfide bonds in target proteins. Recent progress in proteome analysis has resulted in identification of a wide range of potential target proteins...... for Trx, indicating that Trx plays a key role in several aspects of cell metabolism. In contrast to other organisms, plants contain multiple forms of Trx that are classified based on their primary structures and sub-cellular localization. The reduction of cytosolic and mitochondrial types of Trx...

  16. Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

    Science.gov (United States)

    Huang, D Y; Ichikawa, Y

    1997-03-07

    Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic

  17. Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels

    DEFF Research Database (Denmark)

    Gopal, Sandeep; Søgaard, Pernille; Multhaupt, Hinke A B

    2015-01-01

    show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior. In fibroblasts, ligand interactions with heparan sulfate of syndecan-4 recruit cytoplasmic protein kinase C to target serine714 of TRPC7...... with subsequent control of the cytoskeleton and the myofibroblast phenotype. In epidermal keratinocytes a syndecan-TRPC4 complex controls adhesion, adherens junction composition, and early differentiation in vivo and in vitro. In Caenorhabditis elegans, the TRPC orthologues TRP-1 and -2 genetically complement...

  18. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    Energy Technology Data Exchange (ETDEWEB)

    Moriya, Takashi J., E-mail: takashi.moriya@ipmu.jp [Kavli Institute for the Physics and Mathematics of the Universe, Todai Institutes for Advanced Study, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8583 (Japan)

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  19. Meiotic recombination in human oocytes.

    Directory of Open Access Journals (Sweden)

    Edith Y Cheng

    2009-09-01

    Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

  20. [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.

  1. Methods of use of cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  2. Methods of detection using a cellulose binding domain fusion product

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1999-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  3. Electric hydrogen recombiner special tests

    International Nuclear Information System (INIS)

    Wilson, J.F.

    1975-12-01

    Westinghouse has produced an electric hydrogen recombiner to control hydrogen levels in reactor containments following a postulated loss-of-coolant accident. The recombiner underwent extensive testing for NRC qualification (see WCAP 7709-L and Supplements 1, 2, 3, 4). As a result, WCAP 7709-L and Supplements 1, 2, 3, and 4 have been accepted by the NRC for reference in applications not committed to IEEE-323-1974. Supplement 5 and the next supplement will demonstrate conformance to IEEE-323-1974. This supplement describes additional tests, beyond those necessary to qualify the system, which will be referenced in supplement 6. Each test has demonstrated a considerable margin of safety over required performance. Concurrently, the test results increased the fund of technical information on the electric hydrogen recombiner

  4. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

    Science.gov (United States)

    Gutensohn, Michael; Orlova, Irina; Nguyen, Thuong T H; Davidovich-Rikanati, Rachel; Ferruzzi, Mario G; Sitrit, Yaron; Lewinsohn, Efraim; Pichersky, Eran; Dudareva, Natalia

    2013-08-01

    Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  5. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

    Science.gov (United States)

    Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

    2015-06-23

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

  6. A Plant Phytosulfokine Peptide Initiates Auxin-Dependent Immunity through Cytosolic Ca2+ Signaling in Tomato.

    Science.gov (United States)

    Zhang, Huan; Hu, Zhangjian; Lei, Cui; Zheng, Chenfei; Wang, Jiao; Shao, Shujun; Li, Xin; Xia, Xiaojian; Cai, Xinzhong; Zhou, Jie; Zhou, Yanhong; Yu, Jingquan; Foyer, Christine H; Shi, Kai

    2018-03-01

    Phytosulfokine (PSK) is a disulfated pentapeptide that is an important signaling molecule. Although it has recently been implicated in plant defenses to pathogen infection, the mechanisms involved remain poorly understood. Using surface plasmon resonance and gene silencing approaches, we showed that the tomato ( Solanum lycopersicum ) PSK receptor PSKR1, rather than PSKR2, functioned as the major PSK receptor in immune responses. Silencing of PSK signaling genes rendered tomato more susceptible to infection by the economically important necrotrophic pathogen Botrytis cinerea Analysis of tomato mutants defective in either defense hormone biosynthesis or signaling demonstrated that PSK-induced immunity required auxin biosynthesis and associated defense pathways. Here, using aequorin-expressing tomato plants, we provide evidence that PSK perception by tomato PSKR1 elevated cytosolic [Ca 2+ ], leading to auxin-dependent immune responses via enhanced binding activity between calmodulins and the auxin biosynthetic YUCs. Thus, our data demonstrate that PSK acts as a damage-associated molecular pattern and is perceived mainly by PSKR1, which increases cytosolic [Ca 2+ ] and activates auxin-mediated pathways that enhance immunity of tomato plants to B. cinerea . © 2018 American Society of Plant Biologists. All rights reserved.

  7. Nitrate Activation of Cytosolic Protein Kinases Diverts Photosynthetic Carbon from Sucrose to Amino Acid Biosynthesis

    Science.gov (United States)

    Champigny, Marie-Louise; Foyer, Christine

    1992-01-01

    The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis. PMID:16653003

  8. Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

    Science.gov (United States)

    Železnik, Tajana Zajc; Kadin, Andrey; Turk, Vito; Dolenc, Iztok

    2015-09-18

    Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

    Science.gov (United States)

    Lee, Soh-Hyun; Ha, Sun-Ok; Koh, Ho-Jin; Kim, KilSoo; Jeon, Seon-Min; Choi, Myung-Sook; Kwon, Oh-Shin; Huh, Tae-Lin

    2010-02-28

    Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

  10. Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

    Science.gov (United States)

    Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

    2010-04-01

    Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

  11. AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA

    Directory of Open Access Journals (Sweden)

    Yuki Nakaya

    2017-07-01

    Full Text Available Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs that stimulate the induction of interferons (IFNs and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING. Absent in melanoma 2 (AIM2-like receptors (ALRs have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA.

  12. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    International Nuclear Information System (INIS)

    Sherley, J.L.; Kelly, T.J.

    1988-01-01

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity

  13. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Se Jeong [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Gu, Dong Ryun [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Jin, Su Hyun [Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Park, Keun Ha [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Lee, Seoung Hoon, E-mail: leesh2@wku.ac.kr [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Wonkwang Institute of Biomaterials and Implant, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of)

    2016-06-17

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  14. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    International Nuclear Information System (INIS)

    Oh, Se Jeong; Gu, Dong Ryun; Jin, Su Hyun; Park, Keun Ha; Lee, Seoung Hoon

    2016-01-01

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  15. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.

    Directory of Open Access Journals (Sweden)

    Marzia Massignani

    Full Text Available BACKGROUND: Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. PRINCIPAL FINDINGS: We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. SIGNIFICANCE: We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

  16. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    Directory of Open Access Journals (Sweden)

    Zhengyi Liu

    2014-01-01

    Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

  17. Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in vivo.

    Science.gov (United States)

    Tew, W P; Malis, C D; Howard, J E; Lehninger, A L

    1981-01-01

    Synthetic 3-phosphocitrate, an extremely potent inhibitor of calcium phosphate crystallization as determined in a nonbiological physical-chemical assay, has many similarities to a mitochondrial factor that inhibits crystallization of nondiffracting amorphous calcium phosphate. In order to determine whether phosphocitrate can prevent uptake and crystallization of calcium phosphate in mitochondria in vivo, it was administered intraperitoneally to animals given large daily doses of calcium gluconate or parathyroid hormone, a regimen that causes massive accumulation and crystallization of calcium phosphate in the mitochondria and cytosol of renal tubule cells in vivo. Administration of phosphocitrate greatly reduced the net uptake of Ca2+ by the kidneys and prevented the appearance of apatite-like crystalline structures within the mitochondrial matrix and cytosol of renal tubule cells. Phosphocitrate, which is a poor chelator of Ca2+, did not reduce the hypercalcemia induced by either agent. These in vivo observations therefore indicate that phosphocitrate acts primarily at the cellular level to prevent the extensive accumulation of calcium phosphate in kidney cells by inhibiting the mitochondrial accumulation or crystallization of calcium phosphate. Images PMID:6946490

  18. The tobacco carcinogen NNK is stereoselectively reduced by human pancreatic microsomes and cytosols.

    Science.gov (United States)

    Trushin, Neil; Leder, Gerhard; El-Bayoumy, Karam; Hoffmann, Dietrich; Beger, Hans G; Henne-Bruns, Doris; Ramadani, Marco; Prokopczyk, Bogdan

    2008-07-01

    Cigarette smoking increases the risk of cancer of the pancreas. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known environmental compound that induces pancreatic cancer in laboratory animals. Concentrations of NNK are significantly higher in the pancreatic juice of smokers than in that of nonsmokers. The chiral NNK metabolite, (R,S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is itself a potent pancreatic carcinogen in rats. The carcinogenicity of NNAL is related to its stereochemistry; (S)-NNAL is a more potent lung tumorigen in the A/J mouse than is (R)-NNAL. In this study, we determined the potential of the human pancreas to convert NNK into NNAL. Human pancreatic microsomes and cytosols were incubated with [5-(3)H]NNK, and the metabolic products were determined by high-performance liquid chromatography (HPLC). (S)-NNAL was the predominant isomer formed in all cytosolic incubations. In ten microsomal samples, NNAL was formed at an average rate of 3.8 +/- 1.6 pmol/mg/min; (R)-NNAL was the predominant isomer in this group. The average rate of NNAL formation in 18 other microsomal samples was significantly lower, 0.13 +/- 0.12 pmol/mg/min (p < 0.001); (S)-NNAL was the predominant isomer formed in this group. In human pancreatic tissues, there is intraindividual variability regarding the capacity for, and stereoselectivity of, carbonyl reduction of NNK.

  19. Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

    Science.gov (United States)

    Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

    2017-05-15

    Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Catalytic properties of ADAM12 and its domain deletion mutants

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Visse, Robert; Sørensen, Hans Peter

    2008-01-01

    of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits...... restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most...... active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower...

  1. Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

    Science.gov (United States)

    Baldissera, Matheus D; Souza, Carine F; Júnior, Guerino B; Verdi, Camila Marina; Moreira, Karen L S; da Rocha, Maria Izabel U M; da Veiga, Marcelo L; Santos, Roberto C V; Vizzotto, Bruno S; Baldisserotto, Bernardo

    2017-09-01

    Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na + , K + -ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na + , K + -ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

    Science.gov (United States)

    Kojer, Kerstin; Bien, Melanie; Gangel, Heike; Morgan, Bruce; Dick, Tobias P; Riemer, Jan

    2012-01-01

    Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (EGSH) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with EGSH-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of EGSH in the IMS, thus explaining a steady-state EGSH in the IMS which is similar to the cytosol. Moreover, we show that the local EGSH contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells. PMID:22705944

  3. Osteoinductive recombinant silk fusion proteins for bone regeneration.

    Science.gov (United States)

    Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

    2017-02-01

    Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT) 15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data

  4. Production and recombination of gluons

    International Nuclear Information System (INIS)

    Temiraliev, A.T.

    2006-01-01

    Full text: Nonlinear Markov process of parton production has been considered. The Kolmogorov equation is applied for the evolution equation based on the approximation of independent gluons production in every decay act. We introduced a 'crossing' parameter and used the combination relations to obtain nonlinear recombination equation for the evolution of gluon structure function. (author)

  5. Recombinator of hydrogen and oxygen

    International Nuclear Information System (INIS)

    Stejskal, J.; Klein, O.; Scholtz, G.; Schmidt, P.; Olaussson, A.

    1976-01-01

    Improvements are proposed for the well known reactors for the catalytic recombination of hydrogen and oxygen, which should permit this being used in contiuous operation in nuclear reactors (BWRs). The improvements concern the geometric arrangement of gas-inlet and -outlet pipes, the inclination of the axis of the catalyst container and the introduction of remote operation. (UWI) [de

  6. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  7. Recombination in hepatitis C virus.

    Science.gov (United States)

    González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

    2011-10-01

    Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

  8. Posttranslational modifications in human plasma MBL and human recombinant MBL

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

    2007-01-01

    the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

  9. Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

    Science.gov (United States)

    Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

    2015-09-01

    The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia.

  10. Live recombinant BHV/BRSV vaccine

    NARCIS (Netherlands)

    Keil, G.M.; Rijsewijk, F.A.M.

    1998-01-01

    The present invention refers to synthetic Bovine Respiratory Syncytium virus genes. Also the invention relates to live attenuated Bovine Herpesvirus recombinants carrying such synthetic genes. Furthermore, the invention relates to vaccines based on these live attenuated recombinants, for the

  11. Hadron production at RHIC: recombination of quarks

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    We discuss quark recombination applied to the hadronization of a quark gluon plasma. It has been shown that the quark recombination model can explain essential features of hadron production measured in high energy heavy ion collisions.

  12. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

  13. Graded Recombination Layers for Multijunction Photovoltaics

    KAUST Repository

    Koleilat, Ghada I.; Wang, Xihua; Sargent, Edward H.

    2012-01-01

    it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron

  14. Recombinant innovation and endogenous technological transitions

    NARCIS (Netherlands)

    Frenken, K.; Izquierdo, L.R.; Zeppini, P.

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce

  15. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    Science.gov (United States)

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit

    2016-01-01

    Abstract Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  16. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    Furukane, Utaro; Yokota, Toshiaki; Oda, Toshiatsu.

    1978-11-01

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  17. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  18. Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis

    OpenAIRE

    Muratori, L; Cataleta, M; Muratori, P; Lenzi, M; Bianchi, F

    1998-01-01

    Background—Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). 
Aims—Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. 
Patients—Twenty one patients with type 2 AIH were studied. 
Methods—A...

  19. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant...

  20. Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

  1. CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain

    Directory of Open Access Journals (Sweden)

    Mariano A. Ostuni

    2014-11-01

    Full Text Available The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.

  2. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  3. Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

    Science.gov (United States)

    Yang, Eun Sun; Park, Jeen-Woo

    2011-05-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

  4. Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation.

    Science.gov (United States)

    Katoh, Yuki; Tamba, Michiko; Matsuda, Manabu; Kikuchi, Kazuhiro; Okamura, Naomichi

    2018-02-26

    In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP + -dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Regulation of singlet oxygen-induced apoptosis by cytosolic NADP+-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kim, Sun Yee; Lee, Su Min; Tak, Jean Kyoung; Choi, Kyeong Sook; Kwon, Taeg Kyu; Park, Jeen-Woo

    2007-08-01

    Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. Recently, we demonstrated that the control of redox balance and the cellular defense against oxidative damage are the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) through supplying NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPc activity in HL-60 cells regulates singlet oxygen-induced apoptosis. When we examined the protective role of IDPc against singlet oxygen-induced apoptosis with HL-60 cells transfected with the cDNA for mouse IDPc in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPc plays an important protective role in apoptosis of HL-60 cells induced by singlet oxygen.

  6. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.

    Science.gov (United States)

    Chen, Qi; Sun, Lijun; Chen, Zhijian J

    2016-09-20

    The recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor STING. The cGAS-STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. Thus, the cGAS pathway must be properly regulated. Here we review the recent advances in understanding of the cGAS-STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease.

  7. Cytosolic phosphoenolpyruvate carboxykinase is a response gene involved in porcine adipocyte adaptation to heat stress.

    Science.gov (United States)

    Qu, Huan; Ajuwon, Kolapo M

    2018-05-04

    Heat stress (HS) leads to increased lipid storage and expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) in pig adipocytes. However, the importance of PCK1 activation and lipid storage in the adaptive response to HS is unknown. Therefore, in vitro experiments were conducted to investigate the effect of PCK1 inhibition with 3-mercaptopicolinic acid (3MPA) on lipid storage and adipocyte response during HS. In vitro culture of adipocytes under HS (41.0 °C) increased (P cultured adipocytes were less able to induce adaptive responses such as upregulation of HSP70 and triglycerides, and this exacerbated ER stress during HS. Thus, PCK1 may function to alleviate ER stress that occurs during HS.

  8. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

    Science.gov (United States)

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

  9. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    Energy Technology Data Exchange (ETDEWEB)

    Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

    1998-12-31

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

  10. Localization of age-related macular degeneration-associated ARMS2 in cytosol, not mitochondria

    Science.gov (United States)

    Wang, Gaofeng; Spencer, Kylee L.; Court, Brenda L.; Olson, Lana M.; Scott, William K.; Haines, Jonathan L.; Pericak-Vance, Margaret A.

    2010-01-01

    PURPOSE To analyze the relationship between ARMS2 and HTRA1 in the association with age-related macular degeneration (AMD) in an independent case-control dataset, and to investigate the subcellular localization of the ARMS2 protein in an in vitro system. METHOD Two SNPs in ARMS2 and HTRA1 were genotyped in 685 cases and 269 controls by Taqman Assay. Allelic association was tested by a χ2 test. A likelihood ratio test (LRT) of full vs. reduced models was utilized to analyze the interaction between ARMS2 and smoking and HTRA1 and smoking, after adjusting for CFH and age. Immunofluorescence and immunoblot were applied to localize ARMS2 in retinal epithelial ARPE-19 cells and COS7 cell transfected by ARMS2 constructs. RESULT Both significantly associated SNP rs10490924 and rs11200638 (P<0.0001) are in strong linkage disequilibrium (LD) (D′=0.97, r2=0.93) that generates virtually identical association test and odds ratios. In separate logistic regression models the interaction effect for both smoking with ARMS2 and with HTRA1 was not statistically significant. Immunofluorescence and immunoblot show that both endogenous and exogenous ARMS2 are mainly distributed in the cytosol, not the mitochondria. Comparing to wild type, ARMS2 A69S is more likely to be associated with cytoskeleton in COS7 cells. CONCLUSIONS The significant associations in ARMS2 and HTRA1 are with polymorphisms in strong LD that confer virtually identical risks, preventing differentiation at the statistical level. We found that ARMS2 was mainly distributed in the cytosol, not in mitochondrial outer membrane as previously reported, suggesting that ARMS2 may not confer risk to AMD through the mitochondrial pathway. PMID:19255159

  11. Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol

    Science.gov (United States)

    McNamara, AL; Kam, WW-Y; Scales, N; McMahon, SJ; Bennett, JW; Byrne, HL; Schuemann, J; Paganetti, H; Banati, R; Kuncic, Z

    2016-01-01

    Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies ~ 70 eV, substantially lower than that of liquid water ~ 78 eV. Monte Carlo simulations for 10 – 50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of ~ 1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol. PMID:27435339

  12. Expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant.

    Science.gov (United States)

    Kim, Ji Young; Shin, Jae Yong; Kim, Miri; Hann, Seung-Kyung; Oh, Sang Ho

    2012-02-01

    Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2). This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  13. AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA.

    Science.gov (United States)

    Nakaya, Yuki; Lilue, Jingtao; Stavrou, Spyridon; Moran, Eileen A; Ross, Susan R

    2017-07-05

    Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. IMPORTANCE Autoimmune diseases like Aicardi-Goutières syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease. Copyright © 2017 Nakaya et al.

  14. Stereoselective sulfate conjugation of racemic 4-hydroxypropranolol by human and rat liver cytosol

    Energy Technology Data Exchange (ETDEWEB)

    Walle, T.; Walle, U.K. (Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston (USA))

    1991-03-01

    The objective of this study was to determine the stereochemistry of sulfoconjugation of a chiral phenolic amine drug, 4-hydroxypropranolol (HOP), by the human liver. The reaction was catalyzed by the 100,000 g cytosol as the phenolsulfotransferase (PST) enzyme source with PAP35S as the co-substrate. The enantiomers of the intact sulfate conjugate formed, (+)-HOP35S and (-)-HOP35S, were separated by HPLC and measured by liquid scintillation spectrometry. Complex velocity vs. substrate concentration curves were obtained with two peaks of activity, one at 3 microM (high affinity) and one at 500 microM (low affinity). The high-affinity reaction demonstrated a high degree of stereoselectivity. Whereas the affinity of the enantiomers for this reaction was identical, with a very low apparent KM value of 0.59 microM, the apparent Vmax value for (+)-HOPS formation was 4.6-fold higher than for (-)-HOPS. In sharp contrast, the low-affinity reaction, with an apparent KM of 65 microM, was not stereoselective. Inhibition of the high-affinity reaction by elevated temperature, but not by dichloronitrophenol, indicated that this activity was due to a monoamine form of PST. Inhibition of the low-affinity reaction by dichloronitrophenol, but not by elevated temperature, indicated that this activity was due to a phenol form of PST. As a comparison, experiments with the rat liver cytosol demonstrated only one activity, with apparent KM values of 50 microM for both enantiomers and opposite stereoselectivity in maximum velocity compared to humans, {plus minus}-HOPS ratio 0.72. The results of this study demonstrate stereoselectivity in human hepatic sulfation of a chiral phenolic amine, with clear differences between PST isoenzymes.

  15. Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

    Science.gov (United States)

    Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

    2011-07-01

    The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  16. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Takeuchi

    Full Text Available Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.

  17. Development and characterization of recombinant ovine coagulation factor VIII.

    Directory of Open Access Journals (Sweden)

    Philip M Zakas

    Full Text Available Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate

  18. Recombination Catalysts for Hypersonic Fuels

    Science.gov (United States)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  19. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Nakai, Sayaka; Machida, Isamu; Tsuji, Satsuki

    1985-01-01

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G 2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  20. Interface recombination influence on carrier transport

    International Nuclear Information System (INIS)

    Konin, A

    2013-01-01

    A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

  1. Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    OpenAIRE

    Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

    2003-01-01

    Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

  2. Identification and characterization of B-cell epitopes in the DBL4e domain of VAR2CSA

    DEFF Research Database (Denmark)

    Ditlev, Sisse B; Nielsen, Morten A; Resende, Mafalda

    2012-01-01

    and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4e domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4e peptide-array to identify epitopes targeted by DBL4e...... might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4e domain....

  3. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  4. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  5. Consequences of recombination on traditional phylogenetic analysis

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

  6. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... the autophosphorylation site. It is suggested that the acidic domain of the beta subunit, encompassing residues 55-71, plays a role in the interactions between the beta and alpha subunits....

  7. Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

    NARCIS (Netherlands)

    Visch, H.J.; Koopman, W.J.H.; Zeegers, D.; Emst-de Vries, S.E. van; Kuppeveld, F.J.M. van; Heuvel, L.W. van den; Smeitink, J.A.M.; Willems, P.H.G.M.

    2006-01-01

    Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial

  8. Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase

    DEFF Research Database (Denmark)

    Chen, Yun; Zhang, Yiming; Siewers, Verena

    2015-01-01

    Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported...

  9. Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora

    2006-01-01

    Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic...

  10. The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol

    Directory of Open Access Journals (Sweden)

    Flores Rhonda

    2011-04-01

    Full Text Available Abstract Background The periplasmic High Temperature Requirement protein A (HtrA plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA in chlamydial pathogenesis is not clear. Results The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor. Conclusion Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

  11. Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies.

    Science.gov (United States)

    Korzeniewski, Bernard; Zoladz, Jerzy A

    2002-07-01

    Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

  12. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  13. Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

    NARCIS (Netherlands)

    Herberts, Carla A.; Neijssen, Joost J.; de Haan, Jolanda; Janssen, Lennert; Drijfhout, Jan Wouter; Reits, Eric A.; Neefjes, Jacques J.

    2006-01-01

    Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are

  14. Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

    Science.gov (United States)

    Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

    2017-11-17

    With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

  15. Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

    Science.gov (United States)

    Stukenbrock, Eva H; Dutheil, Julien Y

    2018-03-01

    Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

  16. HIGH-n HYDROGEN RECOMBINATION LINES FROM THE FIRST GALAXIES

    International Nuclear Information System (INIS)

    Rule, E.; Loeb, A.; Strelnitski, V. S.

    2013-01-01

    We investigate the prospects of blind and targeted searches in the radio domain (10 MHz to 1 THz) for high-n hydrogen recombination lines from the first generation of galaxies, at z ∼ 4 km s –1 , allow us to assess the blind search time necessary for detection by a given facility. We show that the chances for detection are the highest in the millimeter and submillimeter domains, but finding spontaneous emission in a blind search, especially from redshifts z >> 1, is a challenge even with powerful facilities, such as the Actama Large Millimeter/Submillimeter Array and Square Kilometre Array. The probability of success is higher for a targeted search of lines with principal quantum number n ∼ 10 in Lyman-break galaxies amplified by gravitational lensing. Detection of more than one hydrogen line in such a galaxy will allow for line identification and a precise determination of the galaxy's redshift

  17. PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration▿ †

    Science.gov (United States)

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-01-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development. PMID:17933907

  18. PaTrx1 and PaTrx3, two cytosolic thioredoxins of the filamentous ascomycete Podospora anserina involved in sexual development and cell degeneration.

    Science.gov (United States)

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-12-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development.

  19. Cloning, Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1 in a Highly Salt Tolerant Mangrove (Sonneratia alba

    Directory of Open Access Journals (Sweden)

    Enze Yang

    2015-12-01

    Full Text Available Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1 cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1. SaCSD1 comprised a complete open reading frame (ORF of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90% with the superoxide dismutase (CSD of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO, glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS, H2O2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.

  20. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  1. Faster and stronger manifestation of mitochondrial diseases in skeletal muscle than in heart related to cytosolic inorganic phosphate (Pi) accumulation.

    Science.gov (United States)

    Korzeniewski, Bernard

    2016-08-01

    A model of the cell bioenergetic system was used to compare the effect of oxidative phosphorylation (OXPHOS) deficiencies in a broad range of moderate ATP demand in skeletal muscle and heart. Computer simulations revealed that kinetic properties of the system are similar in both cases despite the much higher mitochondria content and "basic" OXPHOS activity in heart than in skeletal muscle, because of a much higher each-step activation (ESA) of OXPHOS in skeletal muscle than in heart. Large OXPHOS deficiencies lead in both tissues to a significant decrease in oxygen consumption (V̇o2) and phosphocreatine (PCr) and increase in cytosolic ADP, Pi, and H(+) The main difference between skeletal muscle and heart is a much higher cytosolic Pi concentration in healthy tissue and much higher cytosolic Pi accumulation (level) at low OXPHOS activities in the former, caused by a higher PCr level in healthy tissue (and higher total phosphate pool) and smaller Pi redistribution between cytosol and mitochondria at OXPHOS deficiency. This difference does not depend on ATP demand in a broad range. A much greater Pi increase and PCr decrease during rest-to-moderate work transition in skeletal muscle at OXPHOS deficiencies than at normal OXPHOS activity significantly slows down the V̇o2 on-kinetics. Because high cytosolic Pi concentrations cause fatigue in skeletal muscle and can compromise force generation in skeletal muscle and heart, this system property can contribute to the faster and stronger manifestation of mitochondrial diseases in skeletal muscle than in heart. Shortly, skeletal muscle with large OXPHOS deficiencies becomes fatigued already during low/moderate exercise. Copyright © 2016 the American Physiological Society.

  2. Synthesis and characterization of recombinant abductin-based proteins.

    Science.gov (United States)

    Su, Renay S-C; Renner, Julie N; Liu, Julie C

    2013-12-09

    Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

  3. Copper Coordination in the Full-Length, Recombinant Prion Protein†

    Science.gov (United States)

    Burns, Colin S.; Aronoff-Spencer, Eliah; Legname, Giuseppe; Prusiner, Stanley B.; Antholine, William E.; Gerfen, Gary J.; Peisach, Jack; Millhauser, Glenn L.

    2010-01-01

    The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991–4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760–13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92–96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin–echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion. PMID:12779334

  4. Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

    Directory of Open Access Journals (Sweden)

    Spaan Willy JM

    2009-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. Results A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH. The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. Conclusion Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

  5. Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD+-NADH redox potentials

    Directory of Open Access Journals (Sweden)

    Marques-da-Silva A.C.

    1997-01-01

    Full Text Available The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools; gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100

  6. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    Science.gov (United States)

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  7. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    International Nuclear Information System (INIS)

    Fraenkel, A.H.M.

    1974-01-01

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  8. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

    Science.gov (United States)

    Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

    2016-01-01

    Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

  9. Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs

    International Nuclear Information System (INIS)

    Yasuda, Shin; Idell, Steven; Fu Jian; Carter, Glendora; Snow, Rhodora; Liu, M.-C.

    2007-01-01

    The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [ 35 S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [ 35 S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [ 35 S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previously prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17β-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC 50 values determined for the sulfation of 17β-estradiol by SULT1A1 were 11.8 μM, 28.2 μM, and 500 μM, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17β-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [ 35 S]sulfated 17β-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17β-estradiol and other endogenous

  10. Effects of deuterated water upon specific activity of some marker enzymes for cytosol and plasmatic membrane

    International Nuclear Information System (INIS)

    Buzgariu, Wanda; Coroiu, Viorica; Moldovan, Lucia; Titescu, G.; Stefanescu, I.

    2004-01-01

    Recently, numerous studies were devoted to the effects of an increased environmental deuterium concentration on physiological characteristics of various biological systems, from monocellular organisms up to mammals. Within these preoccupations the experiments on enzyme activity and parameters are of special interest since they throw light upon the mechanisms in metabolic biochemical reactions (glycolysis, photosynthesis, transport across membranes, etc). The present work concerns the effects of heavy water upon the activity of some enzymes (dehydrogenase-LDH lactate and 5' nucleotidase) implied in different metabolic pathways, serving as functional indicators for some cellular compartments such as the cytosols and cellular membranes. Enzyme activity was determined by growing for 6 days the cells (Hep 2, CHO, fibroblasts) in deuterated culture media at different concentration levels (20%, 40%, 65% si 90%), as well as in a reaction medium deuterated at 99.96%. In case of the first experimental run the LDH activity was monitored for the three cellular lines (Hep 2, CHO, fibroblasts) for different time intervals (1 d, 3 d and 6 d). After the first 24 h of cells' exposure the activity values were similar regardless of the heavy water concentration in the medium. Exposing the cells for longer time (6 days) led to modifications of LDH activity. In contrast to the case of media with relatively moderate D 2 O content, cell growing in conditions of intense deuteration 65% and 90 % D 2 O) led to an increase of cytosolic enzyme activity of about 50%. In case of 5' nucleotidase after 6 days of cell cultivation in deuteration conditions the activity decreased to 50% and 70% from the value corresponding to normal conditions for cell growth. This diminution of the activity was characteristic for the media with 65% and 90% D 2 O. In the second experimental run the activities of dehydrogenase lactate and 5' nucleotidase from the cellular homogenate obtained from cells grown in

  11. Cytosolic and nuclear caspase-8 have opposite impact on survival after liver resection for hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Koschny, Ronald; Schemmer, Peter; Schirmacher, Peter; Ganten, Tom M; Brost, Sylvia; Hinz, Ulf; Sykora, Jaromir; Batke, Emanuela M; Singer, Stephan; Breuhahn, Kai; Stremmel, Wolfgang; Walczak, Henning

    2013-01-01

    An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival. We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival. Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and –R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade. Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy

  12. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  13. Vaccine platform recombinant measles virus.

    Science.gov (United States)

    Mühlebach, Michael D

    2017-10-01

    The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

  14. CRMAGE: CRISPR Optimized MAGE Recombineering

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

  15. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    Science.gov (United States)

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  16. Atomic excitation and recombination in external fields

    International Nuclear Information System (INIS)

    Nayfeh, M.H.; Clark, C.W.

    1985-01-01

    This volume offers a timely look at Rydberg states of atoms in external fields and dielectronic recombination. Each topic provides authoritative coverage, presents a fresh account of a flourishing field of current atomic physics and introduces new opportunities for discovery and development. Topics considered include electron-atom scattering in external fields; observations of regular and irregular motion as exemplified by the quadratic zeeman effect and other systems; Rydberg atoms in external fields and the Coulomb geometry; crossed-field effects in the absorption spectrum of lithium in a magnetic field; precise studies of static electric field ionization; widths and shapes of stark resonances in sodium above the saddle point; studies of electric field effects and barium autoionizing resonances; autoionization and dielectronic recombination in plasma electric microfields; dielectronic recombination measurements on multicharged ions; merged beam studies of dielectronic recombination; Rydberg atoms and dielectronic recombination in astrophysics; and observations on dielectronic recombination

  17. Density dependence of dielectronic recombination in selenium

    International Nuclear Information System (INIS)

    Hagelstein, P.L.; Rosen, M.D.; Jacobs, V.L.

    1986-01-01

    Dielectronic recombination has been found to be the dominant recombination process in the determination of the ionization balance of selenium near the Ne-like sequence under conditions relevant to the exploding-foil EUV laser plasmas. The dielectronic recombination process tends to populate excited levels, and these levels in turn are more susceptible to subsequent excitation and ionization than are the ground-state ions. If one defines an effective recombination rate which includes, in addition to the primary recombination, the subsequent excitation and ionization of the additional excited-state population due to the primary recombination, then this effective recombination rate can be density-sensitive at relatively low electron density. We present results for this effective dielectronic recombination rate at an electron density of 3 x 10/sup 20/ electrons/cm 3 for recombination from Ne-like to Na-like selenium and from F-like to Ne-like selenium. In the former case, the effective recombination rate coefficient is found to be 1.8 x 10/sup -11/ cm 3 /sec at 1.0 keV, which is to be compared with the zero-density value of 2.8 x 10/sup -11/ cm 3 /sec. In the latter case (F-like to Ne-like), the effective recombination rate coefficient is found to be 1.3 x 10/sup -11/ cm 3 /sec, which is substantially reduced from the zero-density result of 3.3 x 10/sup -11/ cm 3 /sec. We have examined the effects of dielectronic recombination on the laser gain of the dominant Ne-like 3p-3s transitions and have compared our results with those presented by Whitten et al. [Phys. Rev. A 33, 2171 (1986)

  18. Supersymmetric domain walls

    NARCIS (Netherlands)

    Bergshoeff, Eric A.; Kleinschmidt, Axel; Riccioni, Fabio

    2012-01-01

    We classify the half-supersymmetric "domain walls," i.e., branes of codimension one, in toroidally compactified IIA/IIB string theory and show to which gauged supergravity theory each of these domain walls belong. We use as input the requirement of supersymmetric Wess-Zumino terms, the properties of

  19. Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

    Science.gov (United States)

    Hashiguchi, Takuyu; Kurogi, Katsuhisa; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Yasuda, Shin; Liu, Ming-Cheh; Suiko, Masahito

    2011-01-01

    Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

  20. Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells.

    Science.gov (United States)

    Nahire, Rahul; Haldar, Manas K; Paul, Shirshendu; Ambre, Avinash H; Meghnani, Varsha; Layek, Buddhadev; Katti, Kalpana S; Gange, Kara N; Singh, Jagdish; Sarkar, Kausik; Mallik, Sanku

    2014-08-01

    Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

    2015-05-15

    High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

  2. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Directory of Open Access Journals (Sweden)

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  3. Cellular defense against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase

    International Nuclear Information System (INIS)

    Jo, Seung-Hee; Lee, So-Hyun; Suk Chun, Hang; Min Lee, Su; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

    2002-01-01

    Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury

  4. Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

    2002-03-29

    Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

  5. Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kim, Sun Yee; Park, Jeen-Woo

    2003-03-01

    Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

  6. Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

    Science.gov (United States)

    Guizzunti, Gianni; Zurzolo, Chiara

    2015-01-01

    We previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death.

  7. Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

    DEFF Research Database (Denmark)

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different...... AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57...

  8. Cytosolic Phospholipase A2 Protein as a Novel Therapeutic Target for Spinal Cord Injury

    Science.gov (United States)

    Liu, Nai-Kui; Deng, Ling-Xiao; Zhang, Yi Ping; Lu, Qing-Bo; Wang, Xiao-Fei; Hu, Jian-Guo; Oakes, Eddie; Bonventre, Joseph V; Shields, Christopher B; Xu, Xiao-Ming

    2014-01-01

    Objective The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). Methods A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. Results SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. Interpretation cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI. PMID:24623140

  9. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    2016-07-01

    Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  10. Lipid-Based Liquid Crystalline Nanoparticles Facilitate Cytosolic Delivery of siRNA via Structural Transformation.

    Science.gov (United States)

    He, Shufang; Fan, Weiwei; Wu, Na; Zhu, Jingjing; Miao, Yunqiu; Miao, Xiaran; Li, Feifei; Zhang, Xinxin; Gan, Yong

    2018-04-11

    RNA interference (RNAi) technology has shown great promise for the treatment of cancer and other genetic disorders. Despite the efforts to increase the target tissue distribution, the safe and effective delivery of siRNA to the diseased cells with sufficient cytosolic transport is another critical factor for successful RNAi clinical application. Here, the constructed lipid-based liquid crystalline nanoparticles, called nano-Transformers, can transform thestructure in the intracellular acidic environment and perform high-efficient siRNA delivery for cancer treatment. The developed nano-Transformers have satisfactory siRNA loading efficiency and low cytotoxicity. Different from the traditional cationic nanocarriers, the endosomal membrane fusion induced by the conformational transition of lipids contributes to the easy dissociation of siRNA from nanocarriers and direct release of free siRNA into cytoplasm. We show that transfection with cyclin-dependent kinase 1 (CDK1)-siRNA-loaded nano-Transformers causes up to 95% reduction of relevant mRNA in vitro and greatly inhibits the tumor growth without causing any immunogenic response in vivo. This work highlights that the lipid-based nano-Transformers may become the next generation of siRNA delivery system with higher efficacy and improved safety profiles.

  11. Predominant Role of Cytosolic Phospholipase A2α in Dioxin-induced Neonatal Hydronephrosis in Mice

    Science.gov (United States)

    Yoshioka, Wataru; Kawaguchi, Tatsuya; Fujisawa, Nozomi; Aida-Yasuoka, Keiko; Shimizu, Takao; Matsumura, Fumio; Tohyama, Chiharu

    2014-01-01

    Hydronephrosis is a common disease characterized by dilation of the renal pelvis and calices, resulting in loss of kidney function in the most severe cases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces nonobstructive hydronephrosis in mouse neonates through upregulation of prostaglandin E2 (PGE2) synthesis pathway consisting of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) by a yet unknown mechanism. We here studied possible involvement of cytosolic phospholipase A2α (cPLA2α) in this mechanism. To this end, we used a cPLA2α-null mouse model and found that cPLA2α has a significant role in the upregulation of the PGE2 synthesis pathway through a noncanonical pathway of aryl hydrocarbon receptor. This study is the first to demonstrate the predominant role of cPLA2α in hydronephrosis. Elucidation of the pathway leading to the onset of hydronephrosis using the TCDD-exposed mouse model will deepen our understanding of the molecular basis of nonobstructive hydronephrosis in humans. PMID:24509627

  12. OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids.

    Science.gov (United States)

    Hornung, Veit; Hartmann, Rune; Ablasser, Andrea; Hopfner, Karl-Peter

    2014-08-01

    Recent discoveries in the field of innate immunity have highlighted the existence of a family of nucleic acid-sensing proteins that have similar structural and functional properties. These include the well-known oligoadenylate synthase (OAS) family proteins and the recently identified OAS homologue cyclic GMP-AMP (cGAMP) synthase (cGAS). The OAS proteins and cGAS are template-independent nucleotidyltransferases that, once activated by double-stranded nucleic acids in the cytosol, produce unique classes of 2'-5'-linked second messenger molecules, which - through distinct mechanisms - have crucial antiviral functions. 2'-5'-linked oligoadenylates limit viral propagation through the activation of the enzyme RNase L, which degrades host and viral RNA, and 2'-5'-linked cGAMP activates downstream signalling pathways to induce de novo antiviral gene expression. In this Progress article, we describe the striking functional and structural similarities between OAS proteins and cGAS, and highlight their roles in antiviral immunity.

  13. Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells.

    Science.gov (United States)

    Lecourieux, David; Mazars, Christian; Pauly, Nicolas; Ranjeva, Raoul; Pugin, Alain

    2002-10-01

    Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.

  14. Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

    Science.gov (United States)

    Ragusa, S; Cambria, M T; Scarpa, M; Di Paolo, M L; Falconi, M; Rigo, A; Cambria, A

    2001-11-01

    The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites. Copyright 2001 Academic Press.

  15. Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

    Science.gov (United States)

    León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

    2017-08-21

    The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

  16. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Reid, I.R.; Civitelli, R.; Avioli, L.V.; Hruska, K.A.

    1988-01-01

    It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH i ) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH i by means of an amiloride-inhibitable Na + -H + exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH i with a half-maximal effect at 10 -11 M. The diacylglycerol analogue, phorbol 12-myristate 13-acetate, increased both pH i and [ 3 H]thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of [ 3 H]thymidine incorporation in the presence of amiloride, even though it did not affect pH i in these circumstances. It is concluded that PTH decreases pH i and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH i may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment

  17. Rapid purification of recombinant histones.

    Science.gov (United States)

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  18. Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo

    Science.gov (United States)

    Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T

    2013-01-01

    All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pHcyto) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pHcyto shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pHcyto. Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤4 s) trains of action potentials but did buffer slow (∼60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca2+ increase upon stimulation, and partial inhibition of the plasma membrane Ca2+-ATPase, a Ca2+/H+ exchanger, attenuated pHcyto shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (∼0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pHcyto shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pHcyto shifts cannot be dismissed as artifacts of ex vivo preparations. PMID:23401611

  19. The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

    Science.gov (United States)

    Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

    2014-10-01

    JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi; Tsuchiya, Eiko [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan); Ueno, Masaru, E-mail: scmueno@hiroshima-u.ac.jp [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan); Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan)

    2015-08-14

    Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant and suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.

  1. Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells.

    Science.gov (United States)

    Yang, Eun Sun; Lee, Su-Min; Park, Jeen-Woo

    2010-07-01

    It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells' vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.

  2. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

    2013-01-01

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

  3. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

  4. Polysaccharide fraction from higher plants which strongly interacts with the cytosolic phosphorylase isozyme. I. Isolation and characterization

    International Nuclear Information System (INIS)

    Yang, Yi; Steup, M.

    1990-01-01

    From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by 14 C-labeling experiments in which the glucosyl transfer from [ 14 C]glucose 1-phosphate to the polysaccharide preparation was monitored

  5. Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

    Science.gov (United States)

    Keller, F; Wiemken, A

    1982-12-01

    The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

  6. The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

    Science.gov (United States)

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

  7. Fully-coupled mathematical modeling of actomyosin-cytosolic two-phase flow in a highly deformable moving Keratocyte cell.

    Science.gov (United States)

    Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

    2018-01-23

    Interaction between intracellular dynamics and extracellular matrix (ECM) generally occurred into very thin fragment of moving cell, namely lamellipodia, enables all movable cells to crawl on ECM. In fast-moving cells such as fish Keratocytes, Lamellipodia including most cell area finds a fan-like shape during migration, with a variety of aspect ratio function of fish type. In this work, our purpose is to present a novel and more complete two-dimensional continuum mathematical model of actomyosin-cytosolic two-phase flow of a self-deforming Keratocyte with circular spreaded to steady fan-like shape. In the new approach, in addition to the two-phase flow of the F-actin and cytosol, the G-actin transport was spatiotemporally modeled. We also for the first time modeled the effect of variable volume fraction of the moving F-actin porous network on solute transport in the cytosolic fluid. Our novel fully-coupled mathematical model provides a better understanding of intracellular dynamics of fast-migrating Keratocytes; such as the F-actin centripetal and cytosolic fountain-like flows, free-active myosin distribution, distribution sequence of the G-actin, F-actin, and myosin, and myosin-induced pressure flied of cytoplasm as well as the map of intracellular forces like myosin contraction and adhesion traction. All these results are qualitatively and quantitatively in good agreement with experimental observations. According to a range of value of parameters used in this model, our steady state of moving Keratocyte finds fan-like shape with the same aspect ratio as wide category of fish Keratocytes. This new model can predict shape of Keratocytes in other range of parameter values. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

    OpenAIRE

    Khalid N. M. Abdelazeem; Yogesh Singh; Florian Lang; Madhuri S. Salker

    2017-01-01

    Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of ...

  9. Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

    Directory of Open Access Journals (Sweden)

    Matthias Hardtke-Wolenski

    Full Text Available BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

  10. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    Science.gov (United States)

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  11. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

    Science.gov (United States)

    Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

    2015-10-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling.

    Science.gov (United States)

    Karpinska, Barbara; Alomrani, Sarah Owdah; Foyer, Christine H

    2017-09-26

    Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction-oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1 , WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Authors.

  13. Low glutathione regulates gene expression and the redox potentials of the nucleus and cytosol in Arabidopsis thaliana.

    Science.gov (United States)

    Schnaubelt, Daniel; Queval, Guillaume; Dong, Yingping; Diaz-Vivancos, Pedro; Makgopa, Matome Eugene; Howell, Gareth; De Simone, Ambra; Bai, Juan; Hannah, Matthew A; Foyer, Christine H

    2015-02-01

    Reduced glutathione (GSH) is considered to exert a strong influence on cellular redox homeostasis and to regulate gene expression, but these processes remain poorly characterized. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities. The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 and -320 mV. Growth in the presence of the GSH-synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately -260 mV. GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECATE), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH-synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO. Inhibition of auxin transport had no effect on organ GSH levels, but exogenous auxin decreased the root GSH pool. We conclude that GSH depletion significantly increases the redox potentials of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses, but not oxidative stress. © 2013 John Wiley & Sons Ltd.

  14. [Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

    Science.gov (United States)

    Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

    2017-04-01

    This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
.

  15. Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers.

    Science.gov (United States)

    Stary, Creed M; Hogan, Michael C

    2016-05-15

    The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role. Copyright © 2016 the American Physiological Society.

  16. Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

    Directory of Open Access Journals (Sweden)

    Donghyun Kim

    2017-07-01

    Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

  17. Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

    Directory of Open Access Journals (Sweden)

    RAÚL VINET

    2009-01-01

    Full Text Available Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs respond to acetylcholine (ACh with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23. The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

  18. Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

    Science.gov (United States)

    Lenzi, M; Manotti, P; Muratori, L; Cataleta, M; Ballardini, G; Cassani, F; Bianchi, F B

    1995-01-01

    Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7797126

  19. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    Science.gov (United States)

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  20. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [Univ. of Georgia, Athens, GA (United States)

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  1. Electron-ion recombination at low energy

    International Nuclear Information System (INIS)

    Andersen, L.H.

    1993-01-01

    The work is based on results obtained with a merged-beams experiment. A beam of electronics with a well characterized density and energy distribution was merged with a fast, monoenergetic ion beam. Results have been obtained for radiative recombination and dielectronic recombination at low relative energies (0 to ∼70eV). The obtained energy resolution was improved by about a factor of 30. High vacuum technology was used to suppress interactions with electrons from the environments. The velocity distribution of the electron beam was determined. State-selective dielectronic-recombination measurements were performable. Recombination processes were studied. The theoretical background for radiative recombination and Kramers' theory are reviewed. The quantum mechanical result and its relation to the semiclassical theory is discussed. Radiative recombination was also measured with several different non-bare ions, and the applicability of the semiclassical theory to non-bare ions was investigated. The use of an effective charge is discussed. For dielectronic recombination, the standard theoretical approach in the isolated resonance and independent-processes approximation is debated. The applicability of this method was tested. The theory was able to reproduce most of the experimental data except when the recombination process was sensitive to couplings between different electronic configurations. The influence of external perturbing electrostatic fields is discussed. (AB) (31 refs.)

  2. Recombination rate plasticity: revealing mechanisms by design

    Science.gov (United States)

    Sefick, Stephen; Rushton, Chase

    2017-01-01

    For over a century, scientists have known that meiotic recombination rates can vary considerably among individuals, and that environmental conditions can modify recombination rates relative to the background. A variety of external and intrinsic factors such as temperature, age, sex and starvation can elicit ‘plastic’ responses in recombination rate. The influence of recombination rate plasticity on genetic diversity of the next generation has interesting and important implications for how populations evolve. Further, many questions remain regarding the mechanisms and molecular processes that contribute to recombination rate plasticity. Here, we review 100 years of experimental work on recombination rate plasticity conducted in Drosophila melanogaster. We categorize this work into four major classes of experimental designs, which we describe via classic studies in D. melanogaster. Based on these studies, we highlight molecular mechanisms that are supported by experimental results and relate these findings to studies in other systems. We synthesize lessons learned from this model system into experimental guidelines for using recent advances in genotyping technologies, to study recombination rate plasticity in non-model organisms. Specifically, we recommend (1) using fine-scale genome-wide markers, (2) collecting time-course data, (3) including crossover distribution measurements, and (4) using mixed effects models to analyse results. To illustrate this approach, we present an application adhering to these guidelines from empirical work we conducted in Drosophila pseudoobscura. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109222

  3. Electron-ion recombination in merged beams

    International Nuclear Information System (INIS)

    Wolf, A.; Habs, D.; Lampert, A.; Neumann, R.; Schramm, U.; Schuessler, T.; Schwalm, D.

    1993-01-01

    Detailed studies of recombination processes between electrons and highly charged ions have become possible by recent improvements of merged-beams experiments. We discuss in particular measurements with stored cooled ion beams at the Test Storage Ring (TSR) in Heidelberg. The cross section of dielectronic recombination was measured with high energy resolution for few-electron systems up to the nuclear charge of Cu at a relative energy up to 2.6 keV. At low energy (∼0.1 eV) total recombination rates of several ions were measured and compared with calculated radiative recombination rates. Laser-stimulated recombination of protons and of C 6+ ions was investigated as a function of the photon energy using visible radiation. Both the total recombination rates and the stimulated recombination spectra indicate that in spite of the short interaction time in merged beams, also collisional capture of electrons into weakly bound levels (related to three-body recombination) could be important

  4. Electronic recombination in some physics problems

    International Nuclear Information System (INIS)

    Guzman, O.

    1988-01-01

    This work is related to calculations of electronic recombination rates, as a function of electronic density, electronic temperature, and ion nuclear charge. Recombination times can be calculated and compared to cooling time, in cooling processes of ion beans by electrons from storage rings. (A.C.A.S.) [pt

  5. Generation of Modified Pestiviruses by Targeted Recombination

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Friis, Martin Barfred; Risager, Peter Christian

    involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome...

  6. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  7. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    Science.gov (United States)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  8. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-Luis; Demain, Arnold L

    2009-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding...

  9. High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes.

    Science.gov (United States)

    O'Rourke, B; Reibel, D K; Thomas, A P

    1990-07-01

    A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca2+ transient from fluorescence images of fura-2-loaded [corrected] cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca2+ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca2+ transient (less than 40 ms) and the time to peak shortening (less than 80 ms). The technique was used to examine the effects of beta-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca2+ transients and contractions of single cardiomyocytes. beta-Adrenoceptor stimulation resulted in pronounced increases in peak Ca2+, maximal rates of rise and decay of Ca2+, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca2+ or the extent of shortening but extended the duration of the Ca2+ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca2+ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.

  10. Botanical Polyphenols Mitigate Microglial Activation and Microglia-Induced Neurotoxicity: Role of Cytosolic Phospholipase A2.

    Science.gov (United States)

    Chuang, Dennis Y; Simonyi, Agnes; Cui, Jiankun; Lubahn, Dennis B; Gu, Zezong; Sun, Grace Y

    2016-09-01

    Microglia play a significant role in the generation and propagation of oxidative/nitrosative stress, and are the basis of neuroinflammatory responses in the central nervous system. Upon stimulation by endotoxins such as lipopolysaccharides (LPS), these cells release pro-inflammatory factors which can exert harmful effects on surrounding neurons, leading to secondary neuronal damage and cell death. Our previous studies demonstrated the effects of botanical polyphenols to mitigate inflammatory responses induced by LPS, and highlighted an important role for cytosolic phospholipase A2 (cPLA2) upstream of the pro-inflammatory pathways (Chuang et al. in J Neuroinflammation 12(1):199, 2015. doi: 10.1186/s12974-015-0419-0 ). In this study, we investigate the action of botanical compounds and assess whether suppression of cPLA2 in microglia is involved in the neurotoxic effects on neurons. Differentiated SH-SY5Y neuroblastoma cells were used to test the neurotoxicity of conditioned medium from stimulated microglial cells, and WST-1 assay was used to assess for the cell viability of SH-SY5Y cells. Botanicals such as quercetin and honokiol (but not cyanidin-3-O-glucoside, 3CG) were effective in inhibiting LPS-induced nitric oxide (NO) production and phosphorylation of cPLA2. Conditioned medium from BV-2 cells stimulated with LPS or IFNγ caused neurotoxicity to SH-SY5Y cells. Decrease in cell viability could be ameliorated by pharmacological inhibitors for cPLA2 as well as by down-regulating cPLA2 with siRNA. Botanicals effective in inhibition of LPS-induced NO and cPLA2 phosphorylation were also effective in ameliorating microglial-induced neurotoxicity. Results demonstrated cytotoxic factors from activated microglial cells to cause damaging effects to neurons and potential use of botanical polyphenols to ameliorate the neurotoxic effects.

  11. PGC-1α accelerates cytosolic Ca2+ clearance without disturbing Ca2+ homeostasis in cardiac myocytes

    International Nuclear Information System (INIS)

    Chen, Min; Wang, Yanru; Qu, Aijuan

    2010-01-01

    Energy metabolism and Ca 2+ handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1α in cardiac Ca 2+ signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1α via adenoviral transduction. Our data shows that overexpressing PGC-1α improved myocyte contractility without increasing the amplitude of Ca 2+ transients, suggesting that myofilament sensitivity to Ca 2+ increased. Interestingly, the decay kinetics of global Ca 2+ transients and Ca 2+ waves accelerated in PGC-1α-expressing cells, but the decay rate of caffeine-elicited Ca 2+ transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), but not Na + /Ca 2+ exchange (NCX) contribute to PGC-1α-induced cytosolic Ca 2+ clearance. Furthermore, PGC-1α induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1α did not disturb cardiac Ca 2+ homeostasis, because SR Ca 2+ load and the propensity for Ca 2+ waves remained unchanged. These data suggest that PGC-1α can ameliorate cardiac Ca 2+ cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1α-calcium handing pathway sheds new light on the role of PGC-1α in the therapy of cardiac diseases.

  12. Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

    Science.gov (United States)

    Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

    2008-06-01

    Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

  13. Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast.

    Science.gov (United States)

    Johnson, Courtney R; Weems, Andrew D; Brewer, Jennifer M; Thorner, Jeremy; McMurray, Michael A

    2015-04-01

    Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its "G interface") cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un-expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its "unfoldase" activity, indicating a latent "holdase" activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases. © 2015 Johnson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. A Comparison of Two Yeast MnSODs: Mitochondrial Saccharomyces cerevisiae versus Cytosolic Candida albicans

    International Nuclear Information System (INIS)

    Sheng, Y.; Cabelli, D.; Stich, T.A.; Barnese, K.; Gralla, E.B.; Cascio, D.; Britt, R.D.; Valentine, J.S.

    2011-01-01

    Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O 2 - ). This behavior limits the amount of H 2 O 2 produced at high [O 2 - ]; its desirability can be explained by the multiple roles of H 2 O 2 in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O 2 - ], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn 3+ species in yeast Mn 3+ SODs, including the well-characterized 5-coordinate Mn 3+ species and a 6-coordinate L-Mn 3+ species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O 2 - ].

  15. Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

    Science.gov (United States)

    Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap; Lee, Soh-Hyun; Ryoo, Zae Young; Chang, Kyu-Tae; Park, Jeen-Woo; Park, Dong-Chan; Song, Byoung J; Veech, Richard L; Song, Hebok; Huh, Tae-Lin

    2004-09-17

    NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

  16. Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

    Science.gov (United States)

    Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

    2002-06-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

  17. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

    Science.gov (United States)

    Dey, Bappaditya; Dey, Ruchi Jain; Cheung, Laurene S; Pokkali, Supriya; Guo, Haidan; Lee, Jong-Hee; Bishai, William R

    2015-04-01

    Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

  18. Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Erin L. Foresman

    2013-01-01

    Full Text Available Superoxide (O2•− contributes to the development of cardiovascular disease. Generation of O2•− occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1 or extracellular SOD (SOD3 to blood vessels would differentially protect against O2•−-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad containing the gene for Escherichia coli β-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for β-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05, whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control but not AdSOD1 (34±4%. We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2•− production in vascular disease.

  19. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    International Nuclear Information System (INIS)

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B.

    1989-01-01

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue

  20. Cytosolic 5′-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis

    Science.gov (United States)

    Rietveld, A; Pye, S R; Mariampillai, K; Benveniste, O; Peeters, M T J; Miller, J A L; Hanna, M G; Machado, P M; Parton, M J; Gheorghe, K R; Badrising, U A; Lundberg, I E; Sacconi, S; Herbert, M K; McHugh, N J; Lecky, B R F; Brierley, C; Hilton-Jones, D; Lamb, J A; Roberts, M E; Cooper, R G; Saris, C G J; Pruijn, G J M; Chinoy, H; van Engelen, B G M

    2017-01-01

    Objectives Autoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. Materials and methods Data from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Results Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. Interpretation Differences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype. PMID:28122761

  1. Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

    Science.gov (United States)

    Lilleker, J B; Rietveld, A; Pye, S R; Mariampillai, K; Benveniste, O; Peeters, M T J; Miller, J A L; Hanna, M G; Machado, P M; Parton, M J; Gheorghe, K R; Badrising, U A; Lundberg, I E; Sacconi, S; Herbert, M K; McHugh, N J; Lecky, B R F; Brierley, C; Hilton-Jones, D; Lamb, J A; Roberts, M E; Cooper, R G; Saris, C G J; Pruijn, G J M; Chinoy, H; van Engelen, B G M

    2017-05-01

    Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  2. The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

    Directory of Open Access Journals (Sweden)

    Cletus A. Wezena

    2017-11-01

    Full Text Available The enzymes glyoxalase 1 and 2 (Glo1 and Glo2 are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development.

  3. O-GlcNAcylation mediates the control of cytosolic phosphoenolpyruvate carboxykinase activity via Pgc1α.

    Directory of Open Access Journals (Sweden)

    Pedro Latorre

    Full Text Available PGC1α is a coactivator of many transcription factors and cytosolic phosphoenolpyruvate carboxykinase (PCK1 is a key enzyme for gluconeogenesis. PGC1α interacts with the transcription factor PPARγ to stimulate PCK1 expression and thus de novo glucose synthesis. These proteins are not only important for central energy metabolism but also for supplying intermediates for other metabolic pathways, including lipidogenesis and protein synthesis and might therefore be important factors in the ethiopathogenesis of metabolic disorders like diabetes but also in other pathologies like cancer. Since polymorphisms in these proteins have been related to some phenotypic traits in animals like pigs and PGC1α G482S polymorphism increases fat deposition in humans, we have investigated the molecular basis of such effects focusing on a commonly studied polymorphism in pig Pgc1α, which changes a cysteine at position 430 (WT of the protein to a serine (C430S. Biochemical analyses show that Pgc1α WT stimulates higher expression of human PCK1 in HEK293T and HepG2 cells. Paradoxically, Pgc1α WT is less stable than Pgc1α p.C430S in HEK293T cells. However, the study of different post-translational modifications shows a higher O-GlcNAcylation level of Pgc1α p.C430S. This higher O-GlcNAcylation level significantly decreases the interaction between Pgc1α and PPARγ demonstrating the importance of post-translational glycosylation of PGC1α in the regulation of PCK1 activity. This, furthermore, could explain at least in part the observed epistatic effects between PGC1α and PCK1 in pigs.

  4. Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger.

    Science.gov (United States)

    Bleichrodt, R; Vinck, A; Krijgsheld, P; van Leeuwen, M R; Dijksterhuis, J; Wösten, H A B

    2013-03-15

    Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10-15 μm s(-1). Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.

  5. PHYSICAL CONTACT BETWEEN HUMAN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS MODULATES CYTOSOLIC AND NUCLEAR CALCIUM HOMEOSTASIS.

    Science.gov (United States)

    Hassan, Ghada S; Jacques, Danielle; D'Orleans-Juste, Pedro; Magder, Sheldon; Bkaily, Ghassan

    2018-05-14

    The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is however no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and/or hVSMCs . Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([Ca2+]c) and nuclear calcium ([Ca2+]n) levels via physical contact and/or factors released by both cell types. Quantitative 3D confocal microscopy for [Ca2+]c and [Ca2+]n measurement was performed in cultured hVECs or hVSMCs or in co-culture of hVECs-hVSMCs. Our results show that: 1) physical contact between hVECs-hVECs or hVSMCs-hVSMCs does not affect [Ca2+]c and [Ca2+]n in these two cell types; 2) physical contact between hVECs and hVSMCs induces a significant increase only of [Ca2+]n of hVECs without affecting the level of [Ca2+]c and [Ca2+]n of hVSMCs; and 3) preconditioned culture medium of hVECs or hVSMCs does not affect [Ca2+]c and [Ca2+]n of both types of cells. We concluded that physical contact between hVECs and hVSMCs only modulates [Ca2+]n in hVECs. The increase of [Ca2+]n in hVECs may modulate nuclear functions that are calcium dependent.

  6. Cytosolic Phospholipase A2-α: A Potential Therapeutic Target for Prostate Cancer

    Science.gov (United States)

    Patel, Manish I.; Singh, Jaskirat; Niknami, Marzieh; Kurek, Caroline; Yao, Mu; Lu, Sasa; Maclean, Fiona; King, Nicholas J.C.; Gelb, Michael H.; Scott, Kieran F.; Russell, Pamela J.; Boulas, John; Dong., Qihan

    2008-01-01

    Purpose Cytosolic Phospholipase A2-α (cPLA2-α) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA2-α in prostate cancer (PC) cell line and tissue and the effect of targeting cPLA2-α in-vitro and in-vivo. Experimental Design The expression of cPLA2-α was determined in PC cells by RT-PCR, Western blot and immunocytochemistry. Growth inhibition, apoptosis and cPLA2-α activity were determined after inhibition with cPLA2-α siRNA or inhibitor (Wyeth-1). cPLA2-α inhibitor or vehicle was also administered to PC xenograft mouse models. Finally the expression of phospho-cPLA2-α was determined by immunohistochemistry in human normal, androgen sensitive and insensitive PC specimens. Results cPLA2-α is present in all PC cells lines, but increased in androgen insensitive cells. Inhibition with siRNA or Wyeth-1 results in significant reductions in PC cell numbers, as a result of reduced proliferation as well as increased apoptosis and this was also associated with a reduction in cPLA2-α activity. Expression of cyclin D1 and phosphorylation of Akt were also observed to decrease. Wyeth-1 inhibited PC3 xenograft growth by approximately 33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phospho-cPLA2-α is increased when hormone refractory is reached. Conclusions cPLA2-α expression and activation is increased in the androgen insensitive cancer cell line and tissue. Inhibition of cPLA2-α results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory PC. PMID:19088022

  7. Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

    Science.gov (United States)

    Neef, Maren; Ecke, Margret; Hampp, Rüdiger

    2015-07-01

    In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca^{2+}_{ {cyt}}) concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8 g and μ g during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca^{2+}_{ {cyt}} level during the hypergravity phases of a parabola but a significant increase of the Ca^{2+}_{ {cyt}} concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca^{2+}_{ {cyt}} response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca^{2+}_{ {cyt}} changes entirely. We thus conclude that the significant increase in Ca^{2+}_{ {cyt}} under microgravity is largely due to extracellular Ca2+ sources.

  8. Conserved Domain Database (CDD)

    Data.gov (United States)

    U.S. Department of Health & Human Services — CDD is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins.

  9. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

    1997-01-01

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  10. Scedosporium boydii CatA1 and SODC recombinant proteins, new tools for serodiagnosis of Scedosporium infection of patients with cystic fibrosis.

    Science.gov (United States)

    Mina, Sara; Staerck, Cindy; Marot, Agnès; Godon, Charlotte; Calenda, Alphonse; Bouchara, Jean-Philippe; Fleury, Maxime J J

    2017-12-01

    Scedosporium species rank the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), after Aspergillus fumigatus. In CF, these fungi may cause various respiratory infections similar to those caused by A. fumigatus, including bronchitis and allergic broncho-pulmonary mycoses. Diagnosis of these infections relies on the detection of serum antibodies using crude antigenic extracts. However, many components of these extracts are common to Scedosporium and Aspergillus species, leading to cross-reactions. Here, 5 recombinant proteins from S. apiospermum or S. boydii were produced, and their value in serodiagnosis of Scedosporium infections was investigated by enzyme-linked immunosorbent assay. Two of them, corresponding to the Scedosporium catalase A1 or cytosolic Cu,Zn-superoxyde dismutase, allowed the detection of Scedosporium infection, and the differentiation with an Aspergillus infection. These recombinant proteins therefore may serve as a basis for the development of a standardized serological test. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....

  12. BIOTECHNOLOGY OF RECOMBINANT HORMONES IN DOPING

    Directory of Open Access Journals (Sweden)

    Biljana Vitošević

    2011-09-01

    Full Text Available Recombinant DNA technology has allowed rapid progress in creating biosynthetic gene products for the treatment of many diseases. In this way it can produce large amounts of hormone, which is intended for the treatment of many pathological conditions. Recombinant hormones that are commonly used are insulin, growth hormone and erythropoietin. Precisely because of the availability of these recombinant hormones, it started their abuse by athletes. Experiments in animal models confirmed the potential effects of some of these hormones in increasing physical abilities, which attracted the attention of athletes who push the limits of their competitive capability by such manipulation. The risks of the use of recombinant hormones in doping include serious consequences for the health of athletes. Methods of detection of endogenous hormones from recombined based on the use of a monoclonal antibodies, capillary zone electrophoresis and protein biomarkers

  13. Effects of UV radiation on genetic recombination

    International Nuclear Information System (INIS)

    Vlahovic, K.; Zahradka, D.; Petranovic, M.; Petranovic, D.

    1996-01-01

    We have used the model consisting of Escherichia coli cells and l phage to study the effects of UV radiation on genetic recombination. We found two radiation induced processes that reduce or inhibit genetic recombination. One such process leads to the inability of prophage to excise itself from the irradiated bacterial chromosome by the site-specific recombination. The other process was shown to inhibit a type of general recombination by which the prophage transfers one of its genetic markers to the infecting homologous phage. Loss of the prophage ability to take part in both site-specific and general recombination was shown to develop in recB + but not in recB cells. From this we infer that the loss of prophage recombinogenicity in irradiated cells is a consequence of one process in which RecBCD enzyme (the product of recB, recC and recD genes) plays an essential role. (author)

  14. Particle correlations in the recombination model associated with modified Kuti-Weisskopf structure functions

    International Nuclear Information System (INIS)

    Takasugi, E.; Tata, X.

    1982-01-01

    The recombination model associated with modified Kuti-Weisskopf multiquark structure functions is used to analyze particle production by hadronic collisions. The justification of the use of the impulse approximation in these processes and the universal nature of the recombination process are discussed. Single-meson inclusive production in the fragmentation domains of the proton, the pion, and the kaon is used as an input to determine the primitive structure functions. Our parameter-free predictions for low-p/sub T/ multimeson and associated meson-baryon inclusive production are found to be in good agreement with a large amount of recently obtained correlation data. It is pointed out, however, that reactions involving multivalence recombination fall outside the scope of present considerations

  15. Membrane Localization is Critical for Activation of the PICK1 BAR Domain

    Science.gov (United States)

    Madsen, Kenneth L.; Eriksen, Jacob; Milan-Lobo, Laura; Han, Daniel S.; Niv, Masha Y.; Ammendrup-Johnsen, Ina; Henriksen, Ulla; Bhatia, Vikram K.; Stamou, Dimitrios; Sitte, Harald H.; McMahon, Harvey T.; Weinstein, Harel; Gether, Ulrik

    2013-01-01

    The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment. PMID:18466293

  16. Containment air circulation for optimal hydrogen recombination

    International Nuclear Information System (INIS)

    Spinks, N.; Krause, M.

    1997-01-01

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  17. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    International Nuclear Information System (INIS)

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans

    2014-01-01

    Pyrin domains (PYDs) recruit downstream effector molecules in NLR signalling. A specific charge-relay system suggests a the formation of a signalling complex involving a PYD dimer. The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed

  18. Designer genes. Recombinant antibody fragments for biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Wu, A.M.; Yazaki, P.J. [Beckman Research Institute of the City of Hope, Duarte, CA (United States). Dept. of Molecular Biology

    2000-09-01

    Monoclonal antibodies (MAbs), with high specificity and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C{sub H}3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering

  19. Designer genes. Recombinant antibody fragments for biological imaging

    International Nuclear Information System (INIS)

    Wu, A.M.; Yazaki, P.J.

    2000-01-01

    Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C H 3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and

  20. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  1. The unconventional xer recombination machinery of Streptococci/Lactococci

    NARCIS (Netherlands)

    Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

    Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving

  2. The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

    Directory of Open Access Journals (Sweden)

    Emily Qualls-Creekmore

    2017-07-01

    Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

  3. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    Science.gov (United States)

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  4. Sterol-induced Dislocation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Endoplasmic Reticulum Membranes into the Cytosol through a Subcellular Compartment Resembling Lipid Droplets*

    Science.gov (United States)

    Hartman, Isamu Z.; Liu, Pingsheng; Zehmer, John K.; Luby-Phelps, Katherine; Jo, Youngah; Anderson, Richard G. W.; DeBose-Boyd, Russell A.

    2010-01-01

    Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets. PMID:20406816

  5. Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell...... death and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labelled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining...... a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/ [lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD...

  6. Voltage-sensing phosphatase modulation by a C2 domain

    Directory of Open Access Journals (Sweden)

    Paul M. Castle

    2015-04-01

    Full Text Available The voltage-sensing phosphatase (VSP is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD, the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5P2 (Kalli et al., 2014. Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  7. Voltage-sensing phosphatase modulation by a C2 domain.

    Science.gov (United States)

    Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

    2015-01-01

    The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  8. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  9. Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast.

    Science.gov (United States)

    Fukuda, Tomoyuki; Ohya, Yoshikazu; Ohta, Kunihiro

    2007-10-01

    Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called "recombination cold domains". Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE-VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.

  10. Spreading of a prion domain from cell-to-cell by vesicular transport in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Carmen I Nussbaum-Krammer

    2013-03-01

    Full Text Available Prion proteins can adopt self-propagating alternative conformations that account for the infectious nature of transmissible spongiform encephalopathies (TSEs and the epigenetic inheritance of certain traits in yeast. Recent evidence suggests a similar propagation of misfolded proteins in the spreading of pathology of neurodegenerative diseases including Alzheimer's or Parkinson's disease. Currently there is only a limited number of animal model systems available to study the mechanisms that underlie the cell-to-cell transmission of aggregation-prone proteins. Here, we have established a new metazoan model in Caenorhabditis elegans expressing the prion domain NM of the cytosolic yeast prion protein Sup35, in which aggregation and toxicity are dependent upon the length of oligopeptide repeats in the glutamine/asparagine (Q/N-rich N-terminus. NM forms multiple classes of highly toxic aggregate species and co-localizes to autophagy-related vesicles that transport the prion domain from the site of expression to adjacent tissues. This is associated with a profound cell autonomous and cell non-autonomous disruption of mitochondrial integrity, embryonic and larval arrest, developmental delay, widespread tissue defects, and loss of organismal proteostasis. Our results reveal that the Sup35 prion domain exhibits prion-like properties when expressed in the multicellular organism C. elegans and adapts to different requirements for propagation that involve the autophagy-lysosome pathway to transmit cytosolic aggregation-prone proteins between tissues.

  11. Expression, refolding and spectroscopic characterization of fibronectin type III (FnIII)-homology domains derived from human fibronectin leucine rich transmembrane protein (FLRT)-1,-2, and-3

    DEFF Research Database (Denmark)

    Yang, Lila; Falkesgaard, Maria Hansen; Thulstrup, Peter Waaben

    2017-01-01

    various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains...... that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a Kd for each domain, all Kds being in the µM range....

  12. Electron-ion recombination rates for merged-beams experiments

    International Nuclear Information System (INIS)

    Pajek, M.

    1994-01-01

    Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

  13. First-principles study of Frenkel pair recombination in tungsten

    International Nuclear Information System (INIS)

    Qin, Shi-Yao; Jin, Shuo; Li, Yu-Hao; Zhou, Hong-Bo; Zhang, Ying; Lu, Guang-Hong

    2017-01-01

    The recombination of one Frenkel pair in tungsten has been investigated through first-principles simulation. Two different recombination types have been identified: instantaneous and thermally activated. The small recombination barriers for thermally activated recombination cases indicate that recombination can occur easily with a slightly increased temperature. For both of the two recombination types, recombination occurs through the self-interstitial atom moving towards the vacancy. The recombination process can be direct or through replacement sequences, depending on the vertical distance between the vacancy and the 〈1 1 1〉 line of self-interstitial atom pair.

  14. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  15. Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry

    Science.gov (United States)

    Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibo...

  16. Induction of homologous recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, J R; Moore, P D

    1988-09-01

    We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

  17. Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

    Science.gov (United States)

    Liebsch, Filip; Aurousseau, Mark R P; Bethge, Tobias; McGuire, Hugo; Scolari, Silvia; Herrmann, Andreas; Blunck, Rikard; Bowie, Derek; Multhaup, Gerd

    2017-08-11

    The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala 463 and Cys 466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

    Science.gov (United States)

    Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

    2018-03-17

    Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

  19. Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain?

    OpenAIRE

    Nisimoto, Yukio; Jackson, Heather M.; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J. David

    2010-01-01

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47 phox and p67 phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K m for NADPH of 55 ? 10 ?M. The concentration of Nox4 in cell lysates was esti...

  20. Expression and Production of SH2 Domain Proteins.

    Science.gov (United States)

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  1. Insights into evolution in Andean Polystichum (Dryopteridaceae) from expanded understanding of the cytosolic phosphoglucose isomerase gene.

    Science.gov (United States)

    Lyons, Brendan M; McHenry, Monique A; Barrington, David S

    2017-07-01

    Cytosolic phosphoglucose isomerase (pgiC) is an enzyme essential to glycolysis found universally in eukaryotes, but broad understanding of variation in the gene coding for pgiC is lacking for ferns. We used a substantially expanded representation of the gene for Andean species of the fern genus Polystichum to characterize pgiC in ferns relative to angiosperms, insects, and an amoebozoan; assess the impact of selection versus neutral evolutionary processes on pgiC; and explore evolutionary relationships of selected Andean species. The dataset of complete sequences comprised nine accessions representing seven species and one hybrid from the Andes and Serra do Mar. The aligned sequences of the full data set comprised 3376 base pairs (70% of the entire gene) including 17 exons and 15 introns from two central areas of the gene. The exons are highly conserved relative to angiosperms and retain substantial homology to insect pgiC, but intron length and structure are unique to the ferns. Average intron size is similar to angiosperms; intron number and location in insects are unlike those of the plants we considered. The introns included an array of indels and, in intron 7, an extensive microsatellite array with potential utility in analyzing population-level histories. Bayesian and maximum-parsimony analysis of 129 variable nucleotides in the Andean polystichums revealed that 59 (1.7% of the 3376 total) were phylogenetically informative; most of these united sister accessions. The phylogenetic trees for the Andean polystichums were incongruent with previously published cpDNA trees for the same taxa, likely the result of rapid evolutionary change in the introns and contrasting stability in the exons. The exons code a total of seven amino-acid substitutions. Comparison of non-synonymous to synonymous substitutions did not suggest that the pgiC gene is under selection in the Andes. Variation in pgiC including two additional accessions represented by incomplete sequences

  2. Multilayer network representation of membrane potential and cytosolic calcium concentration dynamics in beta cells

    International Nuclear Information System (INIS)

    Gosak, Marko; Dolenšek, Jurij; Markovič, Rene; Slak Rupnik, Marjan; Marhl, Marko; Stožer, Andraž

    2015-01-01

    Highlights: • Physiological processes within and among pancreatic beta cells are very complex. • We analyze the simultaneous recordings of membrane potential and calcium dynamics. • We represent the interaction patterns among beta cells as a multilayer network. • The nature of the intracellular dynamics is found to rely on the network structure. - Abstract: Modern theory of networks has been recognized as a very successful methodological concept for the description and analysis of complex systems. However, some complex systems are more complex than others. For instance, several real-life systems are constituted by interdependent subsystems and their elements are subjected to different types of interactions that can also change with time. Recently, the multilayer network formalism has been proposed as a general theoretical framework for the description and analysis of such multi-dimensional complex systems and is acquiring more and more prominence in terms of a new research direction. In the present study, we use this methodology for the description of functional connectivity patterns and signal propagation between pancreatic beta cells in an islet of Langerhans at the levels of membrane potential (MP) and cytosolic calcium concentration ([Ca"2"+]_c) dynamics to study the extent of overlap in the two networks and to clarify whether time lags between the two signals in individual cells are in any way dependent on the role these cells play in the functional networks. The two corresponding network layers are constructed on the basis of signal directions and pairwise correlations, whereas the interlayer connections represent the time lag between both measured signals. Our results confirm our previous finding that both MP and [Ca"2"+]_c change spread across an islet in the form of a depolarization and a [Ca"2"+]_c wave, respectively. Both types of waves follow nearly the same path and the networks in both layers have a similar but not entirely the same structure

  3. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  4. Control of glucokinase translocation in rat hepatocytes by sorbitol and the cytosolic redox state.

    Science.gov (United States)

    Agius, L

    1994-02-15

    In rat hepatocytes cultured in 5 mM glucose, glucokinase activity is present predominantly in a bound state, and during permeabilization of the cells with digitonin in the presence of Mg2+ less than 20% of glucokinase activity is released. However, incubation of hepatocytes with a higher [glucose] [concn. giving half-maximal activation (A50) 15 mM] or with fructose (A50 50 microM) causes translocation of glucokinase from its Mg(2+)-dependent binding site to an alternative site [Agius and Peak (1993) Biochem. J. 296, 785-796]. A comparison of various substrates showed that sorbitol (A50 8 microM) was 6-fold more potent than fructose at causing glucokinase translocation, whereas tagatose was as potent and mannitol was > 10-fold less potent (A50 550 microM). These substrates also stimulate glucose conversion into glycogen with a similar relative potency, suggesting that conversion of glucose into glycogen is dependent on the binding and/or location of glucokinase within the hepatocyte. Ethanol and glycerol inhibited the effects of fructose, sorbitol and glucose on glucokinase translocation, whereas dihydroxy-acetone had a small additive effect at sub-maximal substrate stimulation. The converse effects of glycerol and dihydroxy-acetone suggest a role for the cytosolic NADH/NAD+ redox state in controlling glucokinase translocation. Titrations with three competitive inhibitors of glucokinase did not provide evidence for involvement of glucokinase flux in glucose-induced glucokinase translocation: N-acetylglucosamine inhibited glucose conversion into glycogen, but not glucose-induced glucokinase translocation; glucosamine partially suppressed glucose-induced and fructose-induced glucokinase translocation, at concentrations that caused total inhibition of glucose conversion into glycogen; D-mannoheptulose increased glucokinase release and had an additive effect with glucose. 3,3'-Tetramethylene-glutaric acid (5 mM), an inhibitor of aldose reductase, inhibited glucokinase

  5. Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

    Directory of Open Access Journals (Sweden)

    Remy Froissart

    2005-03-01

    Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

  6. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. Advances in recombinant antibody manufacturing.

    Science.gov (United States)

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

  8. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Science.gov (United States)

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  9. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jongtae; Hong, Seong-Wan [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Gun Hong [Kyungwon E-C Co., Seongnam (Korea, Republic of)

    2014-10-15

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

  10. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    International Nuclear Information System (INIS)

    Kim, Jongtae; Hong, Seong-Wan; Kim, Gun Hong

    2014-01-01

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

  11. Wave-packet approach to Rydberg resonances in dissociative recombination

    International Nuclear Information System (INIS)

    Morisset, Sabine; Pichl, Lukas; Orel, Ann E.; Schneider, Ioan F.

    2007-01-01

    We report the time-dependent approach to resonant electron capture into Rydberg states in collisions with molecular cations at low impact energy, as an alternative to the method based on multichannel quantum defect theory (MQDT), and present the results for the HD + ion. The propagation of the initial wave function on 13 Rydberg states (besides one valence state) correctly describes the indirect dissociative recombination mechanism in the time domain. Notably, the nonlocal coupling operator between the ionization and dissociation channels is accounted for in the indirect process, extending previous work on the case of direct coupling. The present approach compares to the MQDT framework with remarkable precision: resonant structures in the cross section correctly emerge from the wave-packet propagation; the time-dependent result also forms a cross section envelope for the dense series of ultrafine MQDT resonances corresponding to the quasicontinuous part of the Rydberg state manifold

  12. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-05-01

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  13. Laser-induced electron--ion recombination used to study enhanced spontaneous recombination during electron cooling

    International Nuclear Information System (INIS)

    Schramm, U.; Wolf, A.; Schuess ler, T.; Habs, D.; Schwalm, D.; Uwira, O.; Linkemann, J.; Mueller, A.

    1997-01-01

    Spontaneous recombination of highly charged ions with free electrons in merged velocity matched electron and ion beams has been observed in earlier experiments to occur at rates significantly higher than predicted by theoretical estimates. To study this enhanced spontaneous recombination, laser induced recombination spectra were measured both in velocity matched beams and in beams with well defined relative velocities, corresponding to relative electron-ion detuning energies ranging from 1 meV up to 6.5 meV where the spontaneous recombination enhancement was found to be strongly reduced. Based on a comparison with simplified calculations, the development of the recombination spectra for decreasing detuning energies indicates additional contributions at matched velocities which could be related to the energy distribution of electrons causing the spontaneous recombination rate enhancement

  14. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  15. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  16. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  17. Ultramicroscopic observation of recombinant adenoassociated virus ...

    African Journals Online (AJOL)

    Ultramicroscopic observation of recombinant adenoassociated virus type 2 on the surface of formvarcarbon coated copper grids under different relative humidity and incubation time using negative stain transmission electron microscopy.

  18. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  19. New perspectives on recombinant human antibodies

    NARCIS (Netherlands)

    J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

    1996-01-01

    textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

  20. Construction of retroviral recombinant containing human tissue ...

    African Journals Online (AJOL)

    USER

    2010-03-29

    Mar 29, 2010 ... Recombinant retroviral vector containing human tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene was ..... heavy metal ions, the protein could be express in an .... involves adhesion, degradation and movement. To.