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Sample records for major nhej proteins

  1. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

    2008-01-01

    in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar......BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...

  2. DNA double strand break repair in mammalian cells: role of MRE11 and BLM proteins at the initiation of Non Homologous End Joining (NHEJ)

    International Nuclear Information System (INIS)

    Grabarz, Anastazja

    2011-01-01

    DNA double strand breaks (DSBs) are highly cytotoxic lesions, which can lead to genetic rearrangements. Two pathways are responsible for repairing these lesions: homologous recombination (HR) and non homologous end joining (NHEJ). In our laboratory, an intrachromosomal substrate has been established in order to measure the efficiency and the fidelity of NHEJ in living cells (Guirouilh-Barbat 2004). This approach led us to identify a KU-independent alternative pathway, which uses micro homologies in the proximity of the junction to accomplish repair - the alternative NHEJ (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). The goal of my thesis consisted in identifying and characterising major actors of this pathway. In the absence of KU, alternative NHEJ would be initiated by ssDNA resection of damaged ends. We showed that the nuclease activity of MRE11 is necessary for this mechanism. MRE11 overexpression leads to a two fold stimulation of NHEJ efficiency, while the extinction of MRE11 by siRNA results in a two fold decrease. Our results demonstrate that the proteins RAD50 and CtIP act in the same pathway as MRE11. Moreover, in cells deficient for XRCC4, MIRIN - an inhibitor of the MRN complex - leads to a decrease in repair efficiency, implicating MRE11 in alternative NHEJ. We also showed that MRE11 can act in an ATM-dependent and independent manner (Rass et Grabarz Nat Struct Mol Biol 2009). The initiation of break resection needs to be pursued by a more extensive degradation of DNA, which is accomplished in yeast by the proteins Exo1 and Sgs1/Dna2. In human cells, in vitro studies have recently proposed a similar model of a two-step break resection. We chose to elucidate the role of one of the human homologs of Sgs1 - the RecQ helicase BLM - in the resection process. Our experiments show, that he absence of BLM decreases the efficiency of end joining by NHEJ, accompanied by an increase in error-prone events, especially long-range deletions (≥200 nt). This

  3. DNA-PK. The major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway

    International Nuclear Information System (INIS)

    Hashimoto, Mitsumasa; Rao, S.; Tokuno, Osamu; Utsumi, Hiroshi; Takeda, Shunichi

    2003-01-01

    The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia mutated (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H, CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54 -/- ). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs -/-/- ) failed to exhibit wortmannin radiosensitization. On the other hand, severe combined immunodeficiency (SCID) mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM -/- ) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ). (author)

  4. Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

    Science.gov (United States)

    Murray, Jennie E; van der Burg, Mirjam; IJspeert, Hanna; Carroll, Paula; Wu, Qian; Ochi, Takashi; Leitch, Andrea; Miller, Edward S; Kysela, Boris; Jawad, Alireza; Bottani, Armand; Brancati, Francesco; Cappa, Marco; Cormier-Daire, Valerie; Deshpande, Charu; Faqeih, Eissa A; Graham, Gail E; Ranza, Emmanuelle; Blundell, Tom L; Jackson, Andrew P; Stewart, Grant S; Bicknell, Louise S

    2015-03-05

    Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  5. Molecular mechanism of protein assembly on DNA double-strand breaks in the non-homologous end-joining pathway

    International Nuclear Information System (INIS)

    Yano, Ken-ichi; Morotomi-Yano, Keiko; Adachi, Noritaka; Akiyama, Hidenori

    2009-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair. (author)

  6. Widespread Dependence of Backup NHEJ on Growth State: Ramifications for the Use of DNA-PK Inhibitors

    International Nuclear Information System (INIS)

    Singh, Satyendra K.; Wu Wenqi; Zhang Lihua; Klammer, Holger; Wang Minli; Iliakis, George

    2011-01-01

    Purpose: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4 -/- mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. Methods and Materials: DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of γ-H2AX foci, at different stages of growth. Results: Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by γ-H2AX foci analysis, and leads to enhanced cell killing. Conclusions: These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

  7. Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

    Directory of Open Access Journals (Sweden)

    Grégory Hoff

    2016-11-01

    Full Text Available Non homologous end-joining (NHEJ is a double strand break (DSB repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

  8. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Nigel C. Brissett

    2013-11-01

    Full Text Available Nonhomologous end-joining (NHEJ is one of the major DNA double-strand break (DSB repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  9. Major Intrinsic Proteins in Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus

    2010-01-01

    or as sensor devices based on e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix....../separation technology, a unique class of membrane transport proteins is especially interesting the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells...... internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 109 molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other...

  10. Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Pitcher, Robert S; Tonkin, Louise M; Green, Andrew J; Doherty, Aidan J

    2005-08-19

    A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3'-5' exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.

  11. Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

    NARCIS (Netherlands)

    Y.J. Choi (Yong Jun); H. Li (Han); M.Y. Son (Mi Young); X.-H. Wang (Xiao-Hong); J.L. Fornsaglio (Jamie L.); R.W. Sobol (Robert W.); M. Lee (Moonsook); J. Vijg (Jan); S. Imholz (Sandra); M.E.T. Dollé (Martijn); H. van Steeg (Harry); E. Reiling (Erwin); P. Hasty (Paul)

    2014-01-01

    textabstractKu70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB

  12. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Ding, Nan [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Qi, Yongmei; Zhang, Yingmei [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Wang, Jufang [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Huang, Dejun, E-mail: huangdj@lzu.edu.cn [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China)

    2015-09-15

    Highlights: • Cadmium (Cd) exposure delayed the repair of DNA damage induced by X-ray. • Cd exposure altered the phosphorylation of DNA-PKcs on Thr-2609 and Ser-2056 sites. • Cd impaired the formation of XRCC4 and Ligase IV foci, and down-regulated their protein expression. • Zinc mitigated the effects of Cd on DDR by regulating pDNA-PKcs (Thr-2609), XRCC4 and Ligase IV. - Abstract: Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1–8 h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

  13. Major cancer protein amplifies global gene expression

    Science.gov (United States)

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  14. Role of XRCC4 phosphorylation by DNA-PK in the regulation of NHEJ repair pathway of DNA double strand break

    International Nuclear Information System (INIS)

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Kamdar, Radhika P.; Sicheng, Liu; Wanotayan, Rujira; Matsumoto, Yoshihisa

    2014-01-01

    Non-homologous end-joining (NHEJ) is the predominant pathway of DNA double strand breaks in higher eukaryotes and is active throughout the cell cycle. NHEJ repair includes many factors as Ku70/86, DNA-PKcs, XRCC4-Ligase IV complex and XLF (also known as Cernunnos). In these factors, DNA-PKcs acts as central regulator in NHEJ repair. It recruited at the DNA damages site after DNA damage and after association with Ku its kinase activity is activated. It phosphorylates many of important NHEJ proteins in vitro including XRCC4, Ku 70/86, Artemis, and even DNA-PKcs but till now, very less studies have been done to know the role and significance of phosphorylation in the NHEJ repair. Studies by other researchers identified various phosphorylation sites in XRCC4 by DNA-PK using mass spectrometry but these phosphorylation sites were shown to be dispensable for DSB repair. In the present investigation, we identified 3 serine and one new threonine phosphorylation sites in XRCC4 protein by DNA-PK. In vivo phosphorylation at these sites was verified by generating phosphorylation specific antibodies and the requirement for DNA-PK therein was verified by using DNA-PK inhibitor and DNA-PK proficient and deficient cell lines in response to radiation and zeocin treatment. We have also found that phosphorylation at these sites showed dose dependency in response to radiation treatment. The two serine and one threonine phosphorylation site is also biological important as their mutation into alanine significantly elevated radiosensitivity as measured by colony formation assay. Neutral comet assay showed delayed kinetics in DSB repair of these mutants. Furthermore, we have found a protein, with putative DSB repair function, which interacts with domain including the phosphorylation sites.These results indicate that these phosphorylation sites would mediate functional link between XRCC4 and DNA-PK. (author)

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

    International Nuclear Information System (INIS)

    Iliakis, George

    2009-01-01

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

  16. DNA-binding determinants promoting NHEJ by human Polμ.

    Science.gov (United States)

    Martin, Maria Jose; Juarez, Raquel; Blanco, Luis

    2012-12-01

    Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

  17. The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Pengxiang Chang

    2018-02-01

    Full Text Available Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI. Duck enteritis virus (DEV is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ and homology-directed repair (HDR. Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP, and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

  18. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.

    Science.gov (United States)

    Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K; McDevitt, Shane; Kashkina, Ekaterina; Hoang, Trung M; Pomerantz, Richard T; Sfeir, Agnel

    2017-12-01

    Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

  19. Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

    Directory of Open Access Journals (Sweden)

    Pierre Hentges

    2014-12-01

    Full Text Available Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs: homologous recombination (HR and nonhomologous end-joining (NHEJ. DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1 activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

  20. Signature proteins for the major clades of Cyanobacteria

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    Mathews Divya W

    2010-01-01

    Full Text Available Abstract Background The phylogeny and taxonomy of cyanobacteria is currently poorly understood due to paucity of reliable markers for identification and circumscription of its major clades. Results A combination of phylogenomic and protein signature based approaches was used to characterize the major clades of cyanobacteria. Phylogenetic trees were constructed for 44 cyanobacteria based on 44 conserved proteins. In parallel, Blastp searches were carried out on each ORF in the genomes of Synechococcus WH8102, Synechocystis PCC6803, Nostoc PCC7120, Synechococcus JA-3-3Ab, Prochlorococcus MIT9215 and Prochlor. marinus subsp. marinus CCMP1375 to identify proteins that are specific for various main clades of cyanobacteria. These studies have identified 39 proteins that are specific for all (or most cyanobacteria and large numbers of proteins for other cyanobacterial clades. The identified signature proteins include: (i 14 proteins for a deep branching clade (Clade A of Gloebacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a; (ii 5 proteins that are present in all other cyanobacteria except those from Clade A; (iii 60 proteins that are specific for a clade (Clade C consisting of various marine unicellular cyanobacteria (viz. Synechococcus and Prochlorococcus; (iv 14 and 19 signature proteins that are specific for the Clade C Synechococcus and Prochlorococcus strains, respectively; (v 67 proteins that are specific for the Low B/A ecotype Prochlorococcus strains, containing lower ratio of chl b/a2 and adapted to growth at high light intensities; (vi 65 and 8 proteins that are specific for the Nostocales and Chroococcales orders, respectively; and (vii 22 and 9 proteins that are uniquely shared by various Nostocales and Oscillatoriales orders, or by these two orders and the Chroococcales, respectively. We also describe 3 conserved indels in flavoprotein, heme oxygenase and protochlorophyllide oxidoreductase proteins that

  1. Overexpression of the human major vault protein in gangliogliomas.

    NARCIS (Netherlands)

    Aronica, E; Gorter, J.A.; Vliet, van EA; Spliet, WG; Veelen, van CW; Rijen, van PC; Leenstra, S.; Ramkema, MD; Scheffer, G.L.; Scheper, R.J.; Sisodiya, SM; Troost, D.

    2003-01-01

    PURPOSE: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy.

  2. Overexpression of the human major vault protein in gangliogliomas

    NARCIS (Netherlands)

    Aronica, Eleonora; Gorter, Jan A.; van Vliet, Erwin A.; Spliet, Wim G. M.; van Veelen, Cees W. M.; van Rijen, Peter C.; Leenstra, Sieger; Ramkema, Marja D.; Scheffer, George L.; Scheper, Rik J.; Sisodiya, Sanjay M.; Troost, Dirk

    2003-01-01

    Purpose: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy.

  3. Major vault protein in cardiac and smooth muscle.

    Science.gov (United States)

    Shults, Nataliia V; Das, Dividutta; Suzuki, Yuichiro J

    Major vault protein (MVP) is the major component of the vault particle whose functions are not well understood. One proposed function of the vault is to serve as a mechanism of drug transport, which confers drug resistance in cancer cells. We show that MVP can be found in cardiac and smooth muscle. In human airway smooth muscle cells, knocking down MVP was found to cause cell death, suggesting that MVP serves as a cell survival factor. Further, our laboratory found that MVP is S-glutathionylated in response to ligand/receptor-mediated cell signaling. The S-glutathionylation of MVP appears to regulate protein-protein interactions between MVP and a protein called myosin heavy chain 9 (MYH9). Through MYH9 and Vsp34, MVP may form a complex with Beclin-1 that regulates autophagic cell death. In pulmonary vascular smooth muscle, proteasome inhibition promotes the ubiquitination of MVP, which may function as a mechanism of proteasome inhibition-mediated cell death. Investigating the functions and the regulatory mechanisms of MVP and vault particles is an exciting new area of research in cardiovascular/pulmonary pathophysiology.

  4. BAG3: a multifaceted protein that regulates major cell pathways

    Science.gov (United States)

    Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

    2011-01-01

    Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

  5. Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype.

    Science.gov (United States)

    Forrest, Kerrie L; Bhave, Mrinal

    2007-10-01

    The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant-water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry.

  6. Measurement of guinea pig eosinophil major basic protein by radioimmunoassay

    International Nuclear Information System (INIS)

    Wassom, D.L.; Loegering, D.A.; Gleich, G.J.

    1979-01-01

    Guinea pig eosinophil major basic protein (MBP) was measured by radioimmunoassay (RIA) using 131 I-MBP. Two critical features of the assay were: (1) alkylation of the MBP with iodoacetamide prior to radioiodination and (2) inclusion of another basic protein, either protamine or histone, in the phosphate buffer. Freshly isolated non-alkylated MBP was immunologically deficient when compared to alkylated or reduced MBP, but its reactivity could be redtores by reduction with dithiothreitol and alkylation. Reduction and alkylation also restored the immunoreactivity of polymerized MBP. MBP levels were not elevated in sera from guinea pigs parasitized with Trichinella spiralis and having peripheral blood eosinophilia. Muscle extracts from Trichinella infected animals showed significantly higher levels of MBP activity than normal controls. MBP was measurable in extracts of untreated eosinophils, but reduction and alkylation of these extracts increased MBP activity several fold. The RIA permits detection of MBP in body fluids and tissues at levels as low as 2 ng./ml. The RIA is useful in assessing increased or decreased levels of MBP activity in samples from experimental animals when compared to samples from controls. (author)

  7. DNA ligase III is involved in a DNA-PK independent pathway of NHEJ in human cells

    International Nuclear Information System (INIS)

    Wang, H.; Perrault, A.R.; Qin, W.; Wang, H.; Iliakis, G.

    2003-01-01

    Full text: Double strand breaks (DSB) induced by ionizing radiation (IR) and other cytotoxic agents in the genome of higher eukaryotes are thought to be repaired either by homologous recombination repair (HRR), or non-homologous endjoining (NHEJ). We previously reported the operation of two components of NHEJ in vivo: a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK independent component that acts as a backup (basic or B-NHEJ) and operates with kinetics an order of magnitude slower. To gain further insight into the mechanisms of B-NHEJ, we investigated DNA endjoining in extracts 180BR, a human cell line deficient in DNA ligase IV, using an in vitro plasmid-based DNA endjoining assay. An anti DNA ligase III antibody inhibited almost completely DNA endjoining activity in these extracts. On the other hand, an anti DNA ligase I antibody had no measurable effect in DNA endjoining activity. Immunodepletion of DNA ligase III from 180BR cell extracts abolished the DNA endjoining activity, which could be restored by addition of purified human DNA ligase IIIb. Full-length DNA ligase III bound to double stranded DNA and stimulated DNA endjoining in both intermolecular and intramolecular ligation. Furthermore, fractionation of HeLa cell extracts demonstrated the presence of an activity stimulating the function of DNA ligase III. Based on these observations we propose that DNA ligase III is the ligase operating in B-NHEJ

  8. A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

    Science.gov (United States)

    Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

    2008-02-29

    Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

  9. Isolation of Chromatin from Dysfunctional Telomeres Reveals an Important Role for Ring1b in NHEJ-Mediated Chromosome Fusions

    Directory of Open Access Journals (Sweden)

    Cristina Bartocci

    2014-05-01

    Full Text Available When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation.

  10. Species specificity in major urinary proteins by parallel evolution.

    Directory of Open Access Journals (Sweden)

    Darren W Logan

    Full Text Available Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1 diversity, to enable the signaling of multiple behaviors, 2 dynamic regulation, to indicate age and dominance, and 3 species-specificity. Recently, the major urinary proteins (Mups have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues.Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species

  11. A major protein component of the Bacillus subtilis biofilm matrix.

    Science.gov (United States)

    Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

    2006-02-01

    Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

  12. PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF

    Directory of Open Access Journals (Sweden)

    Satish K. Tadi

    2016-10-01

    Full Text Available In mammalian cells, classical non-homologous end joining (c-NHEJ is critical for DNA double-strand break repair induced by ionizing radiation and during V(DJ recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(DJ recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF’s function.

  13. Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

    Science.gov (United States)

    Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

    2011-01-21

    In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    OpenAIRE

    Jette, Nicholas; Lees-Miller, Susan P.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemi...

  15. Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon

    Science.gov (United States)

    Roy, Soumitra; Shirley, Pamela S.; McClelland, Alan; Kaleko, Michael

    1998-01-01

    Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5. PMID:9658137

  16. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    ;1 in Arabidopsis. That led to the discovery that tip4;1 is gametophytic lethal- gene essential for normal seed set. ICP-MS analyses of the elemental composition of tip4;1 heterozygous T-DNA insert mutant plants and 35S::TIP4;1 over-expression plants indicate that AtTIP4;1 has a role in arsenic distribution...... inorganic forms of arsenic in the environment, can be taken up by plants and thus enter the food chain. Once inside the root cells, As(V) is reduced to As(III) which is then extruded to the soil solution or bound to phytochelatins (PCs) and transported to the vacuole in an effort to accomplish...... detoxification. Plant Noduline 26-like Intrinsic Proteins (NIPs) can channel As(III) and consequently influence the detoxification process. The role of the Tonoplast Intrinsic Proteins (TIPs) in As(III) detoxification remains to be clarified, yet TIPs could have an impact on As(III) accumulation in plant cell...

  17. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang, E-mail: songyangwenrong@hotmail.com

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  18. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    NARCIS (Netherlands)

    Nalcacioglu, R.; Marks, H.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an

  19. Longitudinal changes in C-reactive protein, proform of eosinophil major basic protein, and pregnancy-associated plasma protein-A during weight changes in obese children

    DEFF Research Database (Denmark)

    Lausten-Thomsen, Ulrik; Gamborg, Michael; Bøjsøe, Christine

    2015-01-01

    BACKGROUND: Childhood obesity is associated with several complications, including cardiovascular comorbidity. Several biomarkers, such as high-sensitive C-reactive protein (hs-CRP), proform of eosinophil major basic protein (Pro-MBP) and pregnancy associated plasma protein-A (PAPP-A), have equally...

  20. Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

    International Nuclear Information System (INIS)

    Butler, C.A.; Hoffman, P.S.

    1990-01-01

    A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled [(35S]cysteine or [35S]methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus

  1. A family of related proteins is encoded by the major Drosophila heat shock gene family

    International Nuclear Information System (INIS)

    Wadsworth, S.C.

    1982-01-01

    At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites

  2. Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair.

    Directory of Open Access Journals (Sweden)

    Yong Jun Choi

    Full Text Available Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS to repair DNA double strand breaks (DSBs through the nonhomologous end joining (NHEJ pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70(-/- cells and ku80(-/- cells also appeared to have a defect in base excision repair (BER. BER corrects base lesions, apurinic/apyrimidinic (AP sites and single stand breaks (SSBs utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1 and DNA Polymerase β (Pol β. In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80 and/or free Ku80 (not bound to Ku70 possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80(-/- mice had a shorter life span than dna-pkcs(-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT, an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER.

  3. Effects of whey protein and its two major protein components on satiety and food intake in normal-weight women.

    Science.gov (United States)

    Chungchunlam, Sylvia M S; Henare, Sharon J; Ganesh, Siva; Moughan, Paul J

    2017-06-01

    Protein is the most satiating macronutrient and is source dependent, with whey protein thought to be particularly satiating. The purported satiating effect of whey protein may be due to the unique mixture of proteins in whey or to the major constituent individual proteins (β-lactoglobulin and α-lactalbumin). The objective of the study was to compare the effects of isoenergetic (~2100kJ, ~500kcal) preload meals enriched (~50g protein) with either whey protein isolate (WP), β-lactoglobulin (BL) isolate or α-lactalbumin (AL) isolate, on food intake at an ad libitum test meal 120min later and subjective ratings of appetite (hunger, desire to eat, prospective food consumption and fullness) using visual analogue scales (VAS). Twenty adult normal-weight women (mean age 24.2±0.8years; mean BMI 22.7±0.4kg/m 2 ) participated in the study which used a single-blind completely randomised block design, where each subject consumed each of the three preload meals. Energy intake at the ad libitum test meal and total energy intakes (preload+test meal) did not differ between the three preload meals (p>0.05). There were no significant differences observed for the VAS scores and net incremental area under the curve (net iAUC) during the 120min following consumption of the three preload meals for subjective ratings of appetite (p>0.05). The findings show that the satiating effect of whey protein was similar to that of BL or AL individually and suggest that the major whey protein components BL and AL do not mediate the satiating effect of whey protein. The present human trial was registered with the Australian New Zealand Clinical Trials Registry (www.anzctr.org.au) as ACTRN12615000344594. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. siRNAs Targeting Viral Protein 5: The Major Capsid Protein of ...

    African Journals Online (AJOL)

    Purpose: To investigate whether siRNA targeting viral protein 5 (VP5) can become a new treatment for herpes simplex virus type 1 (HSV-1). Methods: Flow cytometry was performed to determine the ratio of siRNA and lipo2000 to reach the highest transfection efficiency. Western blot and q-PCR were performed to determine ...

  5. Homer1a protein expression in schizophrenia, bipolar disorder, and major depression.

    Science.gov (United States)

    Leber, Stefan L; Llenos, Ida C; Miller, Christine L; Dulay, Jeannette R; Haybaeck, Johannes; Weis, Serge

    2017-10-01

    In recent years, there was growing interest in postsynaptic density proteins in the central nervous system. Of the most important candidates of this specialized region are proteins belonging to the Homer protein family. This family of scaffolding proteins is suspected to participate in the pathogenesis of a variety of diseases. The present study aims to compare Homer1a expression in the hippocampus and cingulate gyrus of patients with major psychiatric disorders including schizophrenia, bipolar disorder and major depression. Immunohistochemistry was used to analyze changes of Homer1a protein expression in the hippocampal formation and the cingulate gyrus from the respective disease groups. Glial cells of the cingulate gyrus gray matter showed decreased Homer1a levels in bipolar disorder when compared to controls. The same results were seen when comparing cingulate gyrus gray matter glial cells in bipolar disorder with major depression. Stratum oriens glial cells of the hippocampus showed decreased Homer1a levels in bipolar disorder when compared to controls and major depression. Stratum lacunosum glial cells showed decreased Homer1a levels in bipolar disorder when compared to major depression. In stratum oriens interneurons Homer1a levels were increased in all disease groups when compared to controls. Stratum lucidum axons showed decreased Homer1a levels in bipolar disorder when compared to controls. Our data demonstrate altered Homer1a levels in specific brain regions and cell types of patients suffering from schizophrenia, bipolar disorder and major depression. These findings support the role of Homer proteins as interesting candidates in neuropsychiatric pathophysiology and treatment.

  6. Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein

    International Nuclear Information System (INIS)

    Hackett, R.H.; Setlow, P.

    1988-01-01

    Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins

  7. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  8. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications

    DEFF Research Database (Denmark)

    Halim, Adnan; Carlsson, Michael C; Mathiesen, Caroline Benedicte K

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post...... allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic...

  9. Identification and quantification of major bovine milk proteins by liquid chromatography.

    Science.gov (United States)

    Bordin, G; Cordeiro Raposo, F; de la Calle, B; Rodriguez, A R

    2001-08-31

    In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.

  10. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  11. Major Depression, C-Reactive Protein, and Incident Ischemic Heart Disease in Healthy Men and Women

    NARCIS (Netherlands)

    Surtees, Paul G.; Wainwright, Nicholas W. J.; Boekholdt, S. Matthijs; Luben, Robert N.; Wareham, Nicholas J.; Khaw, Kay-Tee

    2008-01-01

    Objective: To investigate how C-reactive protein (CRP) and major depressive disorder (MDD) relate to each other and to incident ischemic heart disease (IHD). Studies have shown that both depression and raised CRP concentration predict IHD and that elevated CRP is linked with increased risk of

  12. Two major secreted proteins as probiotic effectors of Lactobacillus rhamnosus GG

    NARCIS (Netherlands)

    Claes, I.; Segers, M.; Ossowski, von I.; Reunanen, J.; Palva, A.; Vos, de W.M.

    2011-01-01

    The well-documented probiotic bacterium Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, named Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been previously reported to promote the survival and growth of intestinal epithelial cells. We could demonstrate that

  13. Structural characterization of dioscorin, the major tuber protein of yams, by near infrared Raman spectroscopy

    International Nuclear Information System (INIS)

    Liao, Y-H; Tseng, C-Y; Chen Wenlung

    2006-01-01

    As very little is known about the molecular structure of dioscorin, the major storage protein of yam tuber, we report here FT-Raman spectroscopic investigation of this yam protein isolated from D. alata L., for the first time. According to a series of purification and identification by ion-exchange chromatography, gel chromatography, SDS-PAGE, and MALDI-TOF-MS, it shows that the major storage protein is made up of dioscorin A (M.W. ∼33 kDa) and dioscorin B (M.W. ∼31 kDa). Raman spectral results indicate that the secondary structure of dioscorin A is major in α-helix, while dioscorin B belongs to anti-parallel β- sheet. It also shows that the microenvironment of major amino acids including tyrosine, phenylalanine, tryptophan, and methionine, and cysteine exhibit explicit differences between these two components. The conformation of disulfide bonding in dioscorin A predominates in Gauche-Gauche-Trans form, while Gauche-Gauche-Gauche and Trans-Gauche-Trans share the conformation in dioscorin B. Structural resemblance between dioscorin A and crude yam proteins implies that dioscorin A exhibits structural preference even though its content is lower than dioscorin B

  14. Structural characterization of dioscorin, the major tuber protein of yams, by near infrared Raman spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Y-H [300 University Road, Department of Food Science, National Chiayi University, Chiayi, Taiwan (China); Tseng, C-Y [300 University Road, Department of Food Science, National Chiayi University, Chiayi, Taiwan (China); Chen Wenlung [Department of Chemistry, National Chiayi University, Chiayi, Taiwan (China)

    2006-01-01

    As very little is known about the molecular structure of dioscorin, the major storage protein of yam tuber, we report here FT-Raman spectroscopic investigation of this yam protein isolated from D. alata L., for the first time. According to a series of purification and identification by ion-exchange chromatography, gel chromatography, SDS-PAGE, and MALDI-TOF-MS, it shows that the major storage protein is made up of dioscorin A (M.W. {approx}33 kDa) and dioscorin B (M.W. {approx}31 kDa). Raman spectral results indicate that the secondary structure of dioscorin A is major in {alpha}-helix, while dioscorin B belongs to anti-parallel {beta}- sheet. It also shows that the microenvironment of major amino acids including tyrosine, phenylalanine, tryptophan, and methionine, and cysteine exhibit explicit differences between these two components. The conformation of disulfide bonding in dioscorin A predominates in Gauche-Gauche-Trans form, while Gauche-Gauche-Gauche and Trans-Gauche-Trans share the conformation in dioscorin B. Structural resemblance between dioscorin A and crude yam proteins implies that dioscorin A exhibits structural preference even though its content is lower than dioscorin B.

  15. Structural characterization of dioscorin, the major tuber protein of yams, by near infrared Raman spectroscopy

    Science.gov (United States)

    Liao, Yu-Hsiu; Tseng, Chi-Yin; Chen, Wenlung

    2006-01-01

    As very little is known about the molecular structure of dioscorin, the major storage protein of yam tuber, we report here FT-Raman spectroscopic investigation of this yam protein isolated from D. alata L., for the first time. According to a series of purification and identification by ion-exchange chromatography, gel chromatography, SDS-PAGE, and MALDI-TOF-MS, it shows that the major storage protein is made up of dioscorin A (M.W. ~33 kDa) and dioscorin B (M.W. ~31 kDa). Raman spectral results indicate that the secondary structure of dioscorin A is major in α-helix, while dioscorin B belongs to anti-parallel β- sheet. It also shows that the microenvironment of major amino acids including tyrosine, phenylalanine, tryptophan, and methionine, and cysteine exhibit explicit differences between these two components. The conformation of disulfide bonding in dioscorin A predominates in Gauche-Gauche-Trans form, while Gauche-Gauche-Gauche and Trans-Gauche-Trans share the conformation in dioscorin B. Structural resemblance between dioscorin A and crude yam proteins implies that dioscorin A exhibits structural preference even though its content is lower than dioscorin B.

  16. Constant region of a kappa III immunoglobulin light chain as a major AL-amyloid protein

    DEFF Research Database (Denmark)

    Engvig, J P; Olsen, K E; Gislefoss, R E

    1998-01-01

    AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein and the correspond......AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein...... and the corresponding AL protein as a kappa III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence......-Jones protein. Furthermore, both the Bence-Jones protein and the AL protein were glycosylated, with possibly a glycosylation in the constant part of the light chain....

  17. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    International Nuclear Information System (INIS)

    Nalcacioglu, Remziye; Marks, Hendrik; Vlak, Just M.; Demirbag, Zihni; Oers, Monique M. van

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an immediate-early gene and confirmed that the major capsid protein (MCP) is a late gene. Transcription of DNApol initiated 35 nt upstream and that of MCP 14 nt upstream of the translational start site. In a luciferase reporter gene assay both promoters were active only when cells were infected with CIV. For DNApol sequences between position -27 and -6, relative to the transcriptional start site, were essential for promoter activity. Furthermore, mutation of a G within the sequence TTGTTTT located just upstream of the DNApol transcription initiation site reduced the promoter activity by 25%. Sequences crucial for MCP promoter activity are located between positions -53 and -29

  18. Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.).

    Science.gov (United States)

    Sathe, Shridhar K; Wolf, Walter J; Roux, Kenneth H; Teuber, Suzanne S; Venkatachalam, Mahesh; Sze-Tao, Kar Wai Clara

    2002-07-17

    The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

  19. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

  20. Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

    Directory of Open Access Journals (Sweden)

    Norris-Mullins Brianna

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

  1. ErbB2 regulates NHEJ repair pathway by affecting erbB1-triggered IR-induced Akt activity

    International Nuclear Information System (INIS)

    Toulany, Mahmoud; Peter Rodemann, H.

    2009-01-01

    We have already reported that erbBl-PI3K-AKT signaling is an important pathway in regulating radiation sensitivity and DNA double strand break repair of human tumor cells. In the present study using small interfering RNA and pharmacological inhibitors in non-small cell lung cancer cell lines we investigated the role of Aktl on radiation-induced DNA-PKcs activity and DNA-double strand break (DNA-DSB) repair. Likewise, the function of erbB2 as hetrodimerization partner of erbBl in radiation-induced Akt activity and regulation of DNA-dsb repair through DNA-PKcs was evaluated. In A549 and H460 transfected with AKTl-siRNA radiation-induced phosphorylation of DNA-PKcs the key enzyme regulating NHEJ repair pathway was markedly inhibited. In both cell lines downregulation of Aktl led to a significant enhancement of residual DNA-DSB, i.e. impaired DNA-DSB repair. Interestingly, in cells transfected with DNA-PKcs-siRNA a lack of effect of AKTl-siRNA on enhancement of residual DNA-DSBs was observed. This results indicate that Aktl regulates NHEJ repair in a DNA-PKcs dependent manner

  2. Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses.

    Science.gov (United States)

    Neu, Ursula; Wang, Jianbo; Macejak, Dennis; Garcea, Robert L; Stehle, Thilo

    2011-07-01

    The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.

  3. Dependence of M13 Major Coat Protein Oligomerization and Lateral Segregation of Bilayer Composition

    NARCIS (Netherlands)

    Fernandes, F.; Loura, L.M.S.; Prieto, M.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2003-01-01

    M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or

  4. Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

    International Nuclear Information System (INIS)

    Xiong, Rui; Siegel, David; Ross, David

    2014-01-01

    Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity

  5. Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Rui; Siegel, David; Ross, David, E-mail: david.ross@ucdenver.edu

    2014-10-15

    Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity.

  6. Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major

    International Nuclear Information System (INIS)

    Arakaki, Tracy; Le Trong, Isolde; Phizicky, Eric; Quartley, Erin; DeTitta, George; Luft, Joseph; Lauricella, Angela; Anderson, Lori; Kalyuzhniy, Oleksandr; Worthey, Elizabeth; Myler, Peter J.; Kim, David; Baker, David; Hol, Wim G. J.; Merritt, Ethan A.

    2006-01-01

    The crystal structure of a conserved hypothetical protein from L. major, Pfam sequence family PF04543, structural genomics target ID Lmaj006129AAA, has been determined at a resolution of 1.6 Å. The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (R free = 0.229) at 1.60 Å resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif

  7. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  8. Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

    Science.gov (United States)

    Harder, T C; Harder, M; de Swart, R L; Osterhaus, A D; Liess, B

    1998-04-01

    The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

  9. Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

    Science.gov (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Laurincik, Jozef; Letko, Juraj; Caamaño, Jose Nestor; Day, Billy N; Lai, Liangxue; Prather, Randall S; Sharpe-Timms, Kathy L; Zimmer, Randall; Sutovsky, Miriam

    2005-03-01

    Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

  10. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervous system tumors.

    Science.gov (United States)

    Sasaki, Tsutomu; Hankins, Gerald R; Helm, Gregory A

    2002-01-01

    Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.

  11. Identity of the immunomodulatory proteins from garlic (Allium sativum) with the major garlic lectins or agglutinins.

    Science.gov (United States)

    Clement, Fatima; Pramod, Siddanakoppalu N; Venkatesh, Yeldur P

    2010-03-01

    Garlic (Allium sativum), an important medicinal spice, displays a plethora of biological effects including immunomodulation. Although some immunomodulatory proteins from garlic have been described, their identities are still unknown. The present study was envisaged to isolate immunomodulatory proteins from raw garlic, and examine their effects on certain cells of the immune system (lymphocytes, mast cells, and basophils) in relation to mitogenicity and hypersensitivity. Three protein components of approximately 13 kD (QR-1, QR-2, and QR-3 in the ratio 7:28:1) were separated by Q-Sepharose chromatography of 30 kD ultrafiltrate of raw garlic extract. All the 3 proteins exhibited mitogenic activity towards human peripheral blood lymphocytes, murine splenocytes and thymocytes. The mitogenicity of QR-2 was the highest among the three immunomodulatory proteins. QR-1 and QR-2 displayed hemagglutination and mannose-binding activities; QR-3 showed only mannose-binding activity. Immunoreactivity of rabbit anti-QR-1 and anti-QR-2 polyclonal antisera showed specificity for their respective antigens as well as mutual cross-reactivity; QR-3 was better recognized by anti-QR-2 (82%) than by anti-QR-1 (55%). QR-2 induced a 2-fold higher histamine release in vitro from leukocytes of atopic subjects compared to that of non-atopic subjects. In all functional studies, QR-2 was more potent compared to QR-1. Taken together, all these results indicate that the two major proteins QR-2 and QR-1 present in a ratio of 4:1 in raw garlic contribute to garlic's immunomodulatory activity, and their characteristics are markedly similar to the abundant Allium sativum agglutinins (ASA) I and II, respectively. Copyright 2010 Elsevier B.V. All rights reserved.

  12. Expression of the Major Vault Protein (MVP) and Cellular Vault Particles in Fish.

    Science.gov (United States)

    Margiotta, Alyssa L; Bain, Lisa J; Rice, Charles D

    2017-11-01

    Cellular vaults are ubiquitous 13 mega Da multi-subunit ribonuceloprotein particles that may have a role in nucleocytoplasmic transport. Seventy percent of the vault's mass consists of a ≈100 kDa protein, the major vault protein (MVP). In humans, a drug resistance-associated protein, originally identified as lung resistance protein in metastatic lung cancer, was ultimately shown to be the previously described MVP. In this study, a partial MVP sequence was cloned from channel catfish. Recombinant MVP (rMVP) was used to generate a monoclonal antibody that recognizes full length protein in distantly related fish species, as well as mice. MVP is expressed in fish spleen, liver, anterior kidney, renal kidney, and gills, with a consistent expression in epithelial cells, macrophages, or endothelium at the interface of the tissue and environment or vasculature. We show that vaults are distributed throughout cells of fish lymphoid cells, with nuclear and plasma membrane aggregations in some cells. Protein expression studies were extended to liver neoplastic lesions in Atlantic killifish collected in situ at the Atlantic Wood USA-EPA superfund site on the southern branch of the Elizabeth River, VA. MVP is highly expressed in these lesions, with intense staining at the nuclear membrane, similar to what is known about MVP expression in human liver neoplasia. Additionally, MVP mRNA expression was quantified in channel catfish ovarian cell line following treatment with different classes of pharmacological agents. Notably, mRNA expression is induced by ethidium bromide, which damages DNA. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1981-1992, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies.

    Science.gov (United States)

    Al Shweiki, Mhd Rami; Oeckl, Patrick; Steinacker, Petra; Hengerer, Bastian; Schönfeldt-Lecuona, Carlos; Otto, Markus

    2017-06-01

    Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates. Areas covered: The review includes the human studies found by a PubMed search using the following terms: 'depression cerebrospinal fluid biomarker', 'major depression biomarker CSF', 'depression CSF biomarker', 'proteomics depression', 'proteomics biomarkers in depression', 'proteomics CSF biomarker in depression', and 'major depressive disorder CSF'. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies. Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.

  14. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    Energy Technology Data Exchange (ETDEWEB)

    Perazzolo, Chiara, E-mail: Chiara.Perazzolo@epfl.ch; Verde, Mariachiara [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland); Homans, Steve W. [University of Leeds, Institute of Molecular and Cellular Biology (United Kingdom); Bodenhausen, Geoffrey [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland)

    2007-05-15

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k{sub ex} = 500-2000 s{sup -1} were typically observed in APO-rMUP for residues located adjacent to a {beta}-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change.

  15. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    International Nuclear Information System (INIS)

    Perazzolo, Chiara; Verde, Mariachiara; Homans, Steve W.; Bodenhausen, Geoffrey

    2007-01-01

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k ex = 500-2000 s -1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change

  16. Serodiagnosis of Borrelia miyamotoi disease by measuring antibodies against GlpQ and variable major proteins

    DEFF Research Database (Denmark)

    Koetsveld, J.; Kolyasnikova, N. M.; Wagemakers, A.

    2018-01-01

    previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls. Methods: We assessed seroreactivity against GlpQ and four Vmps in a well-described, longitudinal......, and IgG between 21 and 50 days, after disease onset. Various combinations of GlpQ and Vmps increased sensitivity and/or specificity compared to single antigens. Notably, the GlpQ or variable large protein (Vlp)-15/16 combination yielded a sensitivity of 94.7% (95% CI: 75.4–99.7) 11–20 days after disease......Objectives: Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had...

  17. Antigenic analysis of the major structural protein of the Mason-Pfizer monkey virus

    International Nuclear Information System (INIS)

    Schochetman, G.; Boehm-Truitt, M.; Schlom, J.

    1976-01-01

    The major internal protein, p27 (m.w. 27,000 daltons) of the Mason-Pfizer monkey virus (MPMV) was purified by gel filtration and ion-exchange chromatography and then used to develop a radioimmunoassay (RIA). This RIA was specific for MPMV because no immunologic cross-reactivity was observed between p27 of MPMV and 13 different RNA tumor viruses of mammalian and avian origin. However, the p27 of MPMV grown in three different primate cells exhibited identical antigenic cross-reactivity. In addition, significant levels of p27 were found only in MPMV-infected cells. These results indicate that synthesis of p27 is induced after virus infection and that p27 represents a viral-coded protein

  18. Annotation of Selaginella moellendorffii major intrinsic proteins and the evolution of the protein family in terrestrial plants

    Directory of Open Access Journals (Sweden)

    Hanna Isa Anderberg

    2012-02-01

    Full Text Available Major intrinsic proteins (MIPs also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to six of the seven MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP. Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies.

  19. Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints

    NARCIS (Netherlands)

    Vos, W.L.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2005-01-01

    M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein

  20. Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

    NARCIS (Netherlands)

    Long, G.; Pan, M.; Westenberg, M.; Vlak, J.M.

    2006-01-01

    F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD),

  1. Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis

    Directory of Open Access Journals (Sweden)

    Panthier Jean-Jacques

    2011-01-01

    Full Text Available Abstract Background Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. Results The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively. Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (β and αs1 and the whey acidic protein (WAP, showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11, Csn2 (7 and Wap (8 genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. Conclusion SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence, already identified in casein genes from other species, likely explain the existence of multiple αs1-casein

  2. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

    Directory of Open Access Journals (Sweden)

    Juliana Ide Aoki

    2016-09-01

    Full Text Available Tubercidin (TUB is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.After transfection of a cosmid genomic library into L. major Friedlin (LmjF parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2 containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP. Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER, a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine

  3. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

    Science.gov (United States)

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-09-01

    Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected

  4. Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

    Science.gov (United States)

    Xiao, Chang-Yi; Fu, Bing-Bing; Li, Zhi-Ying; Mushtaq, Gohar; Kamal, Mohammad Amjad; Li, Jia-Hua; Tang, Gui-Cheng; Xiao, Shuo-Shuang

    2015-01-01

    The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

  5. [Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

    Science.gov (United States)

    Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

    2013-03-01

    To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

  6. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    International Nuclear Information System (INIS)

    Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

    2012-01-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO 3 ). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  7. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  8. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

    Science.gov (United States)

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

    2016-02-18

    Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

  9. Interspecies radioimmunoassay for the major structural proteins of primate type-D retroviruses

    International Nuclear Information System (INIS)

    Colcher, D.; Teramoto, Y.A.; Schlom, J.

    1977-01-01

    A competition radioimmunoassay has been developed in which type-D retroviruses from three primate species compete. The assay utilizes the major structural protein (36,000 daltons) of the endogenous squirrel monkey retrovirus and antisera directed against the major structural protein (27,000 daltons) of the Mason-Pfizer monkey virus isolated from rhesus monkeys. Purified preparations of both viruses grown in heterologous cells, as well as extracts of heterologous cells infected with squirrel monkey retrovirus or Mason-Pfizer monkey virus, compete completely in the assay. Addition of an endogenous virus of the langur monkey also results in complete blocking. No blocking in the assay is observed with type-C baboon viruses, woolly monkey virus, and gibbon virus. Various other type-C and type-B viruses also showed no reactivity. An interspecies assay has thus been developed that recognizes the type-D retroviruses from both Old World monkey (rhesus and langur) and New World monkey (squirrel) species

  10. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    International Nuclear Information System (INIS)

    Tang, Xuhua; Hew, Choy Leong

    2007-01-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2 1 2 1 2 1 , with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution

  11. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Xuhua; Hew, Choy Leong, E-mail: dbshewcl@nus.edu.sg [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore)

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  12. G Protein-Linked Signaling Pathways in Bipolar and Major Depressive Disorders

    Directory of Open Access Journals (Sweden)

    Hiroaki eTomita

    2013-12-01

    Full Text Available The G-protein linked signaling system (GPLS comprises a large number of G-proteins, G protein-coupled receptors (GPCRs, GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP, phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD and bipolar disorder (BPD. This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC and anterior cingulate (ACC were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, ‘activated’ cAMP signaling activity in BPD and ‘blunted’ cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.

  13. Molecular cloning of human protein 4.2: A major component of the erythrocyte membrane

    International Nuclear Information System (INIS)

    Sung, L.A.; Chien, Shu; Lambert, K.; Chang, Longsheng; Bliss, S.A.; Bouhassira, E.E.; Nagel, R.L.; Schwartz, R.S.; Rybicki, A.C.

    1990-01-01

    Protein 4.2 (P4.2) comprises ∼5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. The authors now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-air insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of ∼77 and ∼80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates

  14. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  15. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  16. The proform of eosinophil major basic protein: a new maternal serum marker for adverse pregnancy outcome

    DEFF Research Database (Denmark)

    Pihl, Kasper; Larsen, Torben; Rasmussen, Steen

    2009-01-01

    OBJECTIVE: To establish the first trimester serum levels of the proform of eosinophil major basic protein (proMBP) in pregnancies with adverse outcome. Furthermore, to determine the screening performance using proMBP alone and in combination with other first trimester markers. METHODS: A case-control...... study was conducted in a primary hospital setting. The proMBP concentration was measured in cases with small-for-gestational age (SGA) (n = 150), spontaneous preterm delivery (n = 88), preeclampsia (n = 40), gestational hypertension (n = 10) and in controls (n = 500). Concentrations were converted...... to multiples of the median (MoM) in controls and groups were compared using Mann-Whitney U-test. Logistic regression analysis was used to determine significant factors for predicting adverse pregnancy outcome. Screening performance was assessed using receiver operating characteristic curves. RESULTS: The pro...

  17. Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

    International Nuclear Information System (INIS)

    Wilcox, C.A.; Olson, E.N.

    1987-01-01

    The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

  18. Prediction of arsenic and antimony transporter major intrinsic proteins from the genomes of crop plants.

    Science.gov (United States)

    Azad, Abul Kalam; Ahmed, Jahed; Alum, Md Asraful; Hasan, Md Mahbub; Ishikawa, Takahiro; Sawa, Yoshihiro

    2018-02-01

    Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger's positions (FPs)) of all plant MIPs that have been experimentally proven to transport arsenic (As) and antimony (Sb), some substrate-specific signature sequences (SSSS) or specificity determining sites (SDPs) have been predicted. These SSSS or SDPs were determined in 543 MIPs found in the genomes of 12 crop plants; the As and Sb transporters were predicted to be distributed in noduline-26 like intrinsic proteins (NIPs), and every plant had one or several As and Sb transporter NIPs. Phylogenetic grouping of the NIP subfamily based on the ar/R selectivity filter and FPs were linked to As and Sb transport. We further determined the group-wise substrate selectivity profiles of the NIPs in the 12 crop plants. In addition to two NPA regions, the ar/R filter, and FPs, certain amino acids especially in the pore line, loop D, and termini contribute to the functional distinctiveness of the NIP groups. Expression analysis of transcripts in different organs indicated that most of the As and Sb transporter NIPs were expressed in roots. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. [Comparison between hypo- and hyperglucidic diets on protein sparing in major visceral surgery (author's transl)].

    Science.gov (United States)

    Caillard, B; Bourdois, M; Freysz, M; Baguet, G; Laurin, S; Chalmond, B; Desgres, J; Ahouangbevi, A

    1981-01-01

    The authors compare the protein sparing effect of two diets, exclusively intravenous, including the same protein intake, but a different caloric intake, 21 calories/gm nitrogen for diet "A" (20 cases); 138 calories/gm nitrogen for diet "B" (20 cases). This has been observed during the six post-operative days of major visceral surgery: oesophagectomy, total gastrectomy, colic or rectocolic exeresis, sequestrectomy for acute pancreatitis, lots having been drawn for the diets. Daily nitrogen balances have been made and plasmatic and urinary levels of amino-acids have been measured before surgery and on the third and fifth post-operative days. Statistical exploitation is done by variance analysis (linear model of three factors) with a 99% confidence ratio: 1) Patient factor has no influence whatsoever on cumulative nitrogen balance. 2) Time factor arises only on the fourth post-operative day and only in the hypocaloric diet, leading to catabolism. 3) Metabolic condition is determinant. On no cancerous disease, superiority of hypercaloric diet is well demonstrated. On cancerous disease, nitrogen loss is only significantly different on 4th and 5th post-operative day: hypercaloric diet gives a better nitrogen balance.

  20. Oral treponeme major surface protein: Sequence diversity and distributions within periodontal niches.

    Science.gov (United States)

    You, M; Chan, Y; Lacap-Bugler, D C; Huo, Y-B; Gao, W; Leung, W K; Watt, R M

    2017-12-01

    Treponema denticola and other species (phylotypes) of oral spirochetes are widely considered to play important etiological roles in periodontitis and other oral infections. The major surface protein (Msp) of T. denticola is directly implicated in several pathological mechanisms. Here, we have analyzed msp sequence diversity across 68 strains of oral phylogroup 1 and 2 treponemes; including reference strains of T. denticola, Treponema putidum, Treponema medium, 'Treponema vincentii', and 'Treponema sinensis'. All encoded Msp proteins contained highly conserved, taxon-specific signal peptides, and shared a predicted 'three-domain' structure. A clone-based strategy employing 'msp-specific' polymerase chain reaction primers was used to analyze msp gene sequence diversity present in subgingival plaque samples collected from a group of individuals with chronic periodontitis (n=10), vs periodontitis-free controls (n=10). We obtained 626 clinical msp gene sequences, which were assigned to 21 distinct 'clinical msp genotypes' (95% sequence identity cut-off). The most frequently detected clinical msp genotype corresponded to T. denticola ATCC 35405 T , but this was not correlated to disease status. UniFrac and libshuff analysis revealed that individuals with periodontitis and periodontitis-free controls harbored significantly different communities of treponeme clinical msp genotypes (Pdiversity than periodontitis-free controls (Mann-Whitney U-test, Pdiversity of Treponema clinical msp genotypes within their subgingival niches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation

    Science.gov (United States)

    Hurst, Jane L.; Beynon, Robert J.; Armstrong, Stuart D.; Davidson, Amanda J.; Roberts, Sarah A.; Gómez-Baena, Guadalupe; Smadja, Carole M.; Ganem, Guila

    2017-01-01

    When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal. PMID:28337988

  2. The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

    Science.gov (United States)

    Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

    2014-07-15

    In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. The major outer membrane proteins of enterobacteriaceae. Their immunological relatedness and their possible role in bacterial opsonization

    NARCIS (Netherlands)

    Hofstra, Harmen

    1981-01-01

    This thesis deals with immunological investigations of the major outer membrane proteins of the Enterobacteriaceae as a new group of enterobacterial common envelope antigens, and with some aspects of the possible role of antibodies, prepared against these proteins, in host defense mechanisms. ...

  4. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

    NARCIS (Netherlands)

    Claes, I.J.; Schoofs, G.; Regulski, K.; Vos, de W.M.

    2012-01-01

    Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with

  5. Beyond BLASTing: Tertiary and Quaternary Structure Analysis Helps Identify Major Vault Proteins

    Science.gov (United States)

    Daly, Toni K.; Sutherland-Smith, Andrew J.; Penny, David

    2013-01-01

    We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via

  6. Bioinformatic analysis suggests that the Cypovirus 1 major core protein cistron harbours an overlapping gene

    Directory of Open Access Journals (Sweden)

    Atkins John F

    2008-05-01

    Full Text Available Abstract Members of the genus Cypovirus (family Reoviridae are common pathogens of insects. These viruses have linear dsRNA genomes divided into 10–11 segments, which have generally been assumed to be monocistronic. Here, bioinformatic evidence is presented for a short overlapping coding sequence (CDS in the cypovirus genome segment encoding the major core capsid protein VP1, overlapping the 5'-terminal region of the VP1 ORF in the +1 reading frame. In Cypovirus type 1 (CPV-1, a 62-codon AUG-initiated open reading frame (hereafter ORFX is present in all four available segment 1 sequences. The pattern of base variations across the sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP1 reading frame are taken into account; MLOGD software. In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP1. The genomic location of ORFX is consistent with translation via leaky scanning. A 62–64 codon AUG-initiated ORF is present in a corresponding location and reading frame in other available cypovirus sequences (2 CPV-14, 1 CPV-15 and an 87-codon ORFX homologue may also be present in Aedes pseudoscutellaris reovirus. The ORFX amino acid sequences are hydrophilic and basic, with between 12 and 16 Arg/Lys residues in each though, at 7.5–10.2 kDa, the putative ORFX product is too small to appear on typical published protein gels.

  7. Effects of mycobacteria major secretion protein, Ag85B, on allergic inflammation in the lung.

    Directory of Open Access Journals (Sweden)

    Yusuke Tsujimura

    Full Text Available Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B, which is one of the major protein antigens secreted from M. tuberculosis. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.

  8. Structures of two Arabidopsis thaliana major latex proteins represent novel helix-grip folds

    Energy Technology Data Exchange (ETDEWEB)

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, Jr., George N.; Volkman, Brian F.; (MCW); (UW)

    2009-06-02

    Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.

  9. Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

    International Nuclear Information System (INIS)

    Zhang Liyuan; Chen Liesong; Sun Rui; Ji Shengjun; Ding Yanyan; Wu Jia; Tian Ye

    2013-01-01

    Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5 and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0-10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, id est (i.e.) DNA damage caused by 0-10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body's maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body's own ability to repair DNA DSBs via the NHEJ pathway. (author)

  10. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

    Science.gov (United States)

    Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille

    2012-01-01

    Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464

  11. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    Science.gov (United States)

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  12. Major urinary protein (MUP) profiles show dynamic changes rather than individual 'barcode' signatures.

    Science.gov (United States)

    Thoß, M; Luzynski, K C; Ante, M; Miller, I; Penn, D J

    2015-06-30

    House mice ( Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This 'barcode hypothesis' requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent ('major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous ('minor') bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis ('dynamic changes' hypothesis).

  13. Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

    Directory of Open Access Journals (Sweden)

    Sarah L Maguire

    2014-01-01

    Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

  14. Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

    Science.gov (United States)

    Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

    2014-01-01

    In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

  15. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages

    DEFF Research Database (Denmark)

    Jena, Prajna; Mohanty, Soumitra; Mohanty, Tirthankar

    2012-01-01

    Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil...... and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule...... proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane...

  16. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

    Directory of Open Access Journals (Sweden)

    Prajna Jena

    Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

  17. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate.

    Science.gov (United States)

    Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    International Nuclear Information System (INIS)

    Wise, K.S.; Kim, M.F.

    1987-01-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125 I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [ 35 S] methionine, 14 C-amino acids, or [ 3 H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11

  19. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    Energy Technology Data Exchange (ETDEWEB)

    Wise K.S.; Kim, M.F.

    1987-12-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

  20. Distinct radioprotective activities of major heat shock proteins in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Kabakov, Alexander; Malyutina, Yana; Kudryavtsev, Vladimir

    2008-01-01

    Full text: Several years ago we have suggested that heat shock proteins (Hsps) can be involved in cellular and tissue mechanisms of protection from ionizing radiation. At present, the accumulated experimental data do allow us to characterize three major mammalian Hsps, Hsp70, Hsp27 and Hsp90, as specific endogenous radioprotectors which are able to prevent or minimize cell death resulting from the radiation exposure. It follows from the many findings that the radioprotective effect of these Hsps is particularly manifested in their ability to attenuate apoptosis in various normal and tumor cells irradiated in vivo or in vitro. The obtained data already enable to suggest three main mechanisms of the radioprotection conferred by the excess Hsps: 1) Modulation of the intracellular signaling so that the apoptotic signal transduction is blocked, whereas the 'cell survival' signal transduction is stimulated; 2) Suppression of the radiation-associated free radical generation and apoptosis induced by reactive oxygen species (ROS); 3) Attenuation of the genotoxic impact of ionizing radiation. The latter suggested mechanism seems particularly intriguing and implies that the excess Hsps can somehow contribute to protection/repair of genomic DNA from radiation-induced damage. According to our recent results, Hsp90 is indeed involved in the post-irradiation repair of nuclear DNA, while excess Hsp70 can beneficially affect the p53-mediated DNA damage response in irradiated cells to ensure their long-term survival and recovery. As for Hsp27, we found that its accumulation in target cells increases their radioresistance by enhancing the irradiation-responsive activation of anti apoptotic pathways. While the Hsp70 and Hsp27 seem to perform different functions in irradiated cells, the synergistic enhancement of radioprotection was clearly observed in the cells enriched by the both the Hsps. In vivo, such radioprotective activities of the major mammalian Hsps may play a role in

  1. Alteration of major vault protein in human glioblastoma and its relation with EGFR and PTEN status.

    Science.gov (United States)

    Navarro, L; Gil-Benso, R; Megías, J; Muñoz-Hidalgo, L; San-Miguel, T; Callaghan, R C; González-Darder, J M; López-Ginés, C; Cerdá-Nicolás, M J

    2015-06-25

    Glioblastoma (GBM) is the most frequent and malignant primary brain tumor. Conventional therapy of surgical removal, radiation and chemotherapy is largely palliative. Major vault protein (MVP), the main component of the vault organelle has been associated with multidrug resistance by reducing cellular accumulation of chemotherapeutic agents. With regard to cancer, MVP has been shown to be overexpressed in drug resistance development and malignant progression. The aim of the present study was to evaluate the MVP gene dosage levels in 113 archival samples from GBM and its correlation with patients' survival and epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog (PTEN) gene dosages. Fluorescent in situ hybridization revealed polysomy of chromosome 7 in 76.1% of the GBMs and EGFR amplification in a 64.6% of the tumors. Genetic status of EGFR, PTEN and MVP copies was determined by multiplex ligation-dependent probe amplification (MLPA) technique. 31% of the tumors showed the EGFR is variant III mutation (EGFRvIII) mutation and 74.3% of them presented amplification of MVP gene. Amplification of EGFR and MVP was found in a 63.7% and 56.6% of the GBM, respectively. An inverse correlation between MVP and PTEN dosage values was observed. Besides, an inverse relationship between the survival of the patients treated with chemotherapy and the levels of MVP copies was determined. In conclusion, our study reveals an important role of MVP, together with EGFRvIII and PTEN, in the progression of GBM and proposes it as a novel and interesting target for new treatment approaches. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. High C-Reactive Protein Predicts Delirium Incidence, Duration, and Feature Severity After Major Noncardiac Surgery.

    Science.gov (United States)

    Vasunilashorn, Sarinnapha M; Dillon, Simon T; Inouye, Sharon K; Ngo, Long H; Fong, Tamara G; Jones, Richard N; Travison, Thomas G; Schmitt, Eva M; Alsop, David C; Freedman, Steven D; Arnold, Steven E; Metzger, Eran D; Libermann, Towia A; Marcantonio, Edward R

    2017-08-01

    To examine associations between the inflammatory marker C-reactive protein (CRP) measured preoperatively and on postoperative day 2 (POD2) and delirium incidence, duration, and feature severity. Prospective cohort study. Two academic medical centers. Adults aged 70 and older undergoing major noncardiac surgery (N = 560). Plasma CRP was measured using enzyme-linked immunosorbent assay. Delirium was assessed from Confusion Assessment Method (CAM) interviews and chart review. Delirium duration was measured according to number of hospital days with delirium. Delirium feature severity was defined as the sum of CAM-Severity (CAM-S) scores on all postoperative hospital days. Generalized linear models were used to examine independent associations between CRP (preoperatively and POD2 separately) and delirium incidence, duration, and feature severity; prolonged hospital length of stay (LOS, >5 days); and discharge disposition. Postoperative delirium occurred in 24% of participants, 12% had 2 or more delirium days, and the mean ± standard deviation sum CAM-S was 9.3 ± 11.4. After adjusting for age, sex, surgery type, anesthesia route, medical comorbidities, and postoperative infectious complications, participants with preoperative CRP of 3 mg/L or greater had a risk of delirium that was 1.5 times as great (95% confidence interval (CI) = 1.1-2.1) as that of those with CRP less than 3 mg/L, 0.4 more delirium days (P delirium (3.6 CAM-S points higher, P delirium (95% CI = 1.0-2.4) as those in the lowest quartile (≤127.53 mg/L), had 0.2 more delirium days (P delirium (4.5 CAM-S points higher, P delirium incidence, duration, and feature severity. CRP may be useful to identify individuals who are at risk of developing delirium. © 2017, Copyright the Authors Journal compilation © 2017, The American Geriatrics Society.

  3. Effect of caffeine on the expression of a major X-ray induced protein in human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, E.N.; Boothman, D.A. (Univ. of Pennsylvania School of Medicine, Philadelphia (USA))

    1991-03-01

    We have examined the effect of caffeine on the concomitant processes of the repair of potentially lethal damage (PLD) and the synthesis of X-ray-induced proteins in the human malignant melanoma cell line, Ul-Mel. Caffeine administered at a dose of 5mM after X radiation not only inhibited PLD repair but also markedly reduced the level of XIP269, a major X-ray-induced protein whose expression has been shown to correlate with the capacity to repair PLD. The expression of the vast majority of other cellular proteins, including seven other X-ray-induced proteins, remained unchanged following caffeine treatment. A possible role for XIP269 in cell cycle delay following DNA damage by X irradiation is discussed.

  4. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins

    International Nuclear Information System (INIS)

    Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.

    2006-01-01

    For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)

  6. Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees.

    Science.gov (United States)

    Drapeau, Mark David; Albert, Stefan; Kucharski, Robert; Prusko, Carsten; Maleszka, Ryszard

    2006-11-01

    The genomic architecture underlying the evolution of insect social behavior is largely a mystery. Eusociality, defined by overlapping generations, parental brood care, and reproductive division of labor, has most commonly evolved in the Hymenopteran insects, including the honey bee Apis mellifera. In this species, the Major Royal Jelly Protein (MRJP) family is required for all major aspects of eusocial behavior. Here, using data obtained from the A. mellifera genome sequencing project, we demonstrate that the MRJP family is encoded by nine genes arranged in an approximately 60-kb tandem array. Furthermore, the MRJP protein family appears to have evolved from a single progenitor gene that encodes a member of the ancient Yellow protein family. Five genes encoding Yellow-family proteins flank the genomic region containing the genes encoding MRJPs. We describe the molecular evolution of these protein families. We then characterize developmental-stage-specific, sex-specific, and caste-specific expression patterns of the mrjp and yellow genes in the honey bee. We review empirical evidence concerning the functions of Yellow proteins in fruit flies and social ants, in order to shed light on the roles of both Yellow and MRJP proteins in A. mellifera. In total, the available evidence suggests that Yellows and MRJPs are multifunctional proteins with diverse, context-dependent physiological and developmental roles. However, many members of the Yellow/MRJP family act as facilitators of reproductive maturation. Finally, it appears that MRJP protein subfamily evolution from the Yellow protein family may have coincided with the evolution of honey bee eusociality.

  7. Gene divergence of homeologous regions associated with a major seed protein content QTL in soybean

    Directory of Open Access Journals (Sweden)

    Puji eLestari

    2013-06-01

    Full Text Available Understanding several modes of duplication contributing on the present genome structure is getting an attention because it could be related to numerous agronomically important traits. Since soybean serves as a rich protein source for animal feeds and human consumption, breeding efforts in soybean have been directed toward enhancing seed protein content. The publicly available soybean sequences and its genomically featured elements facilitate comprehending of quantitative trait loci (QTL for seed protein content in concordance with homeologous regions in soybean genome. Although parts of chromosome (Chr 20 and Chr 10 showed synteny, QTLs for seed protein content present only on Chr 20. Using comparative analysis of gene contents in recently duplicated genomic regions harboring QTL for protein/oil content on Chrs 20 and 10, a total of 27 genes are present in duplicated regions of both chromosomes. Notably, 4 tandem duplicates of the putative homeobox protein 22 (HB22 are present only on Chr 20 and this Medicago truncatula homolog expressed in endosperm at seed filling stage. These tandem duplicates could contribute on the protein/oil QTL of Chr 20. Our study suggests that non-shared gene contents within the duplicated genomic regions might lead to absence/presence of QTL related to protein/oil content.

  8. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

    Science.gov (United States)

    Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

    2011-11-01

    Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive

  9. Mapping the Interactome of a Major Mammalian Endoplasmic Reticulum Heat Shock Protein 90.

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    Feng Hong

    Full Text Available Up to 10% of cytosolic proteins are dependent on the mammalian heat shock protein 90 (HSP90 for folding. However, the interactors of its endoplasmic reticulum (ER paralogue (gp96, Grp94 and HSP90b1 has not been systematically identified. By combining genetic and biochemical approaches, we have comprehensively mapped the interactome of gp96 in macrophages and B cells. A total of 511 proteins were reduced in gp96 knockdown cells, compared to levels observed in wild type cells. By immunoprecipitation, we found that 201 proteins associated with gp96. Gene Ontology analysis indicated that these proteins are involved in metabolism, transport, translation, protein folding, development, localization, response to stress and cellular component biogenesis. While known gp96 clients such as integrins, Toll-like receptors (TLRs and Wnt co-receptor LRP6, were confirmed, cell surface HSP receptor CD91, TLR4 pathway protein CD180, WDR1, GANAB and CAPZB were identified as potentially novel substrates of gp96. Taken together, our study establishes gp96 as a critical chaperone to integrate innate immunity, Wnt signaling and organ development.

  10. The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

    Directory of Open Access Journals (Sweden)

    Luciana Galetto

    Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

  11. Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

    Directory of Open Access Journals (Sweden)

    Delphine Vincent

    Full Text Available Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs of specific ion series of known proteins and integrate peaks at defined retention time (RT window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

  12. A novel small molecule inhibitor of the DNA repair protein Ku70/80.

    Science.gov (United States)

    Weterings, Eric; Gallegos, Alfred C; Dominick, Lauren N; Cooke, Laurence S; Bartels, Trace N; Vagner, Josef; Matsunaga, Terry O; Mahadevan, Daruka

    2016-07-01

    Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents. Copyright © 2016 Elsevier B.V. All

  13. Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

    Science.gov (United States)

    Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2017-02-01

    Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  15. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans.

    Science.gov (United States)

    Dhondt, Ineke; Petyuk, Vladislav A; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D; Depuydt, Geert; Braeckman, Bart P

    2016-09-13

    Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    Directory of Open Access Journals (Sweden)

    Ineke Dhondt

    2016-09-01

    Full Text Available Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditis elegans and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

  17. Biological activities and applications of dioscorins, the major tuber storage proteins of yam.

    Science.gov (United States)

    Lu, Yeh-Lin; Chia, Cho-Yun; Liu, Yen-Wenn; Hou, Wen-Chi

    2012-01-01

    Yam tubers, a common tuber crop and an important traditional Chinese medicine in Taiwan, have many bioactive substances, including phenolic compounds, mucilage polysaccharides, steroidal saponins and proteins. Among the total soluble proteins, 80% of them are dioscorins. In the past two decades, many studies showed that dioscorins exhibited biological activities both in vitro and in vivo, including the enzymatic, antioxidant, antihypertensive, immunomodulatory, lectin activities and the protecting role on airway epithelial cells against allergens in vitro. Some of these activities are survived after chemical, heating process or enzymatic digestion. Despite of lacking the intact structural information and the detail action mechanisms in the cells, yam dioscorins are potential resources for developing as functional foods and interesting targets for food protein researchers.

  18. Biological Activities and Applications of Dioscorins, the Major Tuber Storage Proteins of Yam

    Directory of Open Access Journals (Sweden)

    Yeh-Lin Lu

    2012-01-01

    Full Text Available Yam tubers, a common tuber crop and an important traditional Chinese medicine in Taiwan, have many bioactive substances, including phenolic compounds, mucilage polysaccharides, steroidal saponins and proteins. Among the total soluble proteins, 80% of them are dioscorins. In the past two decades, many studies showed that dioscorins exhibited biological activities both in vitro and in vivo, including the enzymatic, antioxidant, antihypertensive, immunomodulatory, lectin activities and the protecting role on airway epithelial cells against allergens in vitro. Some of these activities are survived after chemical, heating process or enzymatic digestion. Despite of lacking the intact structural information and the detail action mechanisms in the cells, yam dioscorins are potential resources for developing as functional foods and interesting targets for food protein researchers.

  19. Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.

    Science.gov (United States)

    Verma, Alok Kumar; Sharma, Akanksha; Kumar, Sandeep; Gupta, Rinkesh Kumar; Kumar, Dinesh; Gupta, Kriti; Giridhar, B H; Das, Mukul; Dwivedi, Premendra D

    2016-06-01

    Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to

  20. The porcine acute phase response to infection with Actinobacillus pleuropneumoniae. Haptoglobin, C-reactive protein, major acute phase protein and serum amyloid a protein are sensitive indicators of infection

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Klausen, Joan; Nielsen, J.P.

    1998-01-01

    response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4-5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase......, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility...

  1. Effects of heat shock protein 90 expression on pectoralis major oxidation in broilers exposed to acute heat stress.

    Science.gov (United States)

    Hao, Y; Gu, X H

    2014-11-01

    This study was conducted to determine the effects of heat shock protein 90 (HSP90) expression on pH, lipid peroxidation, heat shock protein 70 (HSP70), and glucocorticoid receptor (GR) expression of pectoralis major in broilers exposed to acute heat stress. In total, 90 male broilers were randomly allocated to 3 groups: control (CON), heat stress (HS), or geldanamycin treatment (GA). On d 41, the broilers in the GA group were injected intraperitoneally with GA (5 μg/kg of BW), and the broilers in the CON and HS groups were injected intraperitoneally with saline. Twenty-four hours later, the broilers in the CON group were moved to environmental chambers controlled at 22°C for 2 h, and the broilers in the HS and GA groups were moved to environmental chambers controlled at 40°C for 2 h. The pH values of the pectoralis major after 30 min and 24 h of chilling after slaughter of HS and GA broilers were significantly lower (P stress caused significant increases in sera corticosterone and lactic dehydrogenase, the activity of malondialdehyde and superoxide dismutase, the expression of HSP90 and HSP70, and nuclear expression of GR protein in the pectoralis major (P stress induced a significant decrease in GR protein expression in the cytoplasm and GR mRNA expression. Furthermore, the low expression of HSP90 significantly increased levels of lactic dehydrogenase and malondialdehyde and GR protein expression in the cytoplasm under heat stress (P shock protein 90 was positively correlated with corticosterone and superoxide dismutase activities (P < 0.01), and HSP90 mRNA was negatively correlated with pH after chilling for 24 h. The results demonstrated that HSP90 plays a pivotal role in protecting cells from oxidation. ©2014 Poultry Science Association Inc.

  2. Leucine is a major regulator of muscle protein synthesis in neonates

    Science.gov (United States)

    Approximately 10 % of infants born in the United States are of low birth weight. Growth failure during the neonatal period is a common occurrence in low birth weight infants due to their inability to tolerate full feeds, concerns about advancing protein supply, and high nutrient requirements for gro...

  3. Major Proteins of the Amyloplast of Agar and Soil - Grown Potato Tubers

    DEFF Research Database (Denmark)

    Hald, Simon; Blennow, Andreas; Stensballe, Allan

    and total tuber extracts by SDS-PAGE and the specific activities of marker enzymes for amyloplast, cytosol, mitochondria and the vacuole. SDS-PAGE separated amyloplast and starch granule proteins were in-gel digested with trypsin, analyzed by mass spectrometry, and identified by searches against presently...

  4. Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    DEFF Research Database (Denmark)

    Juul, Nicolai Stefan; Timmerman, E; Gevaert, K

    2007-01-01

    The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated...

  5. Endocytosis of the major yolk proteins of the silkmoth, Hyalophora cecropia: Uptake kinetics and interactions

    International Nuclear Information System (INIS)

    Kulakosky, P.C.

    1989-01-01

    The oocytes of Lepidopteran insects take up several yolk proteins in defined proportions even though their relative availability in the hemolymph changes during the several days required to complete yolk formation in all the eggs. There are three hemolymph yolk precursors, vitellogenin, microvitellogenin and lipophorin; one precursor, paravitellogenin is produced in the ovary. The control mechanism for their proportional endocytosis is not known. In this thesis, the author describe the purification of all four proteins and the radiolabeling of the hemolymph precursors. The radiolabeled proteins were tested with an in vitro incubation system to assess the biological activity of the proteins and the reliability of the incubation methods. All of the labeled probes were transferred from the incubation medium to yolk spheres within the oocyte in a saturable, energy-dependent, and stage-specific manner. The rates of uptake were similar to the estimated rates of uptake in situ. The concentration dependence of in vitro uptake was investigated and found to be consistent with in situ concentrations and the composition of yolk in mature eggs. Two precursors, vitellogenin and lipophorin, competed for uptake indicating that they share a common binding site while the third, microvitellin, did not compete with the others. Though vitellogenin and lipophorin competed for uptake, only vitellogenin displayed the unique ability to increase the uptake rate of microvitellin and fluid in vitro

  6. SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling

    Czech Academy of Sciences Publication Activity Database

    Dráber, Peter; Vonková, Ivana; Štěpánek, Ondřej; Hrdinka, Matouš; Kucová, Markéta; Skopcová, Tereza; Otáhal, Pavel; Angelisová, Pavla; Hořejší, Václav; Yeung, M.; Weiss, A.; Brdička, Tomáš

    2011-01-01

    Roč. 31, č. 22 (2011), s. 4550-4562 ISSN 0270-7306 R&D Projects: GA MŠk 1M0506; GA ČR GEMEM/09/E011 Institutional research plan: CEZ:AV0Z50520514 Keywords : SCIMP * transmembrane adaptor protein * MHC II Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.527, year: 2011

  7. Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Hubbert, N

    1985-01-01

    or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

  8. Evidence for multiple major histocompatibility class II X-box binding proteins.

    OpenAIRE

    Celada, A; Maki, R

    1989-01-01

    The X box is a loosely conserved DNA sequence that is located upstream of all major histocompatibility class II genes and is one of the cis-acting regulatory elements. Despite the similarity between all X-box sequences, each promoter-proximal X box in the mouse appears to bind a separate nuclear factor.

  9. A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

    Science.gov (United States)

    Osada, Eriko; Akematsu, Takahiko; Asano, Tomoya; Endoh, Hiroshi

    2014-03-01

    Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  10. Investigation and Comparison of Leishmania major Promastigote and Amastigote Protein Content by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    S. Soleimanifard

    2013-04-01

    Full Text Available ntroduction & Objective: Leishmania is a protozoan of the trypanosomatidae family. This pro-tozoan has two stages in its life cycle, promastigote form in sand flies and amastigote form in macrophage of mammalian hosts. The purpose of this study was identification and compari-son of proteins of Leishmania amastigote and promastigote stages. Materials & Methods: The present study is a cross sectional study of two forms of Leishmania major. To culture promastigotes , L.major (MRHO/IR/75/ER from previously infected Balb/c mice was transferred to modified N.N.N medium with overlay of liquid BHI and then transferred to RPMI-1640 at 26oc ± 1 for mass production. After isolation and growth, pro-mastigotes were transferred to liquid cell culture medium RPMI-1640 with pH 5.5 and incu-bated at 5% CO2 at 37oc for 72 hours until promastigote to amastigote transformation. Elec-trophoresis was performed with SDS-PAGE method to find and compare the molecular weight of the antigens of two stages. Results: The molecular weights of the bands observed in both forms were as follows: 19, 36, 50, 63, 65, 80, 90, 94, 96, 110- 130 KDa. The proteins in the surface of only promastigote were 22, 28 and 46 KDa and special proteins in the surface of amastigote were 12 and 32 KDa. Conclusion : According to this study Leishmania parasite has stage specific proteins. Various studies have shown that axenic amastigotes and tissue amastigotes are similar in their protein content. Therefore, based on stage specific proteins ,effective drugs and vaccines can be de-signed against leishmaniasis. (Sci J Hamadan Univ Med Sci 2013; 20 (1:1-8

  11. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    Directory of Open Access Journals (Sweden)

    Michael J Sheehan

    2016-03-01

    Full Text Available Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  12. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  13. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

    Directory of Open Access Journals (Sweden)

    Rebecca Cook

    2015-03-01

    Full Text Available Deficiencies in DNA double-strand break (DSB repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1 is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ. Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.

  14. Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

    DEFF Research Database (Denmark)

    Rognan, D; Lauemoller, S L; Holm, A

    1999-01-01

    A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.......) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation...

  15. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  16. Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

    DEFF Research Database (Denmark)

    Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

    2018-01-01

    Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic but is a c......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...... but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

  17. YB-1 facilitates basal and 5-fluorouracil-inducible expression of the human major vault protein (MVP) gene.

    Science.gov (United States)

    Stein, Ulrike; Bergmann, Stephan; Scheffer, George L; Scheper, Rik J; Royer, Hans-Dieter; Schlag, Peter M; Walther, Wolfgang

    2005-05-19

    Vaults have been suggested to play a direct role in multidrug resistance (MDR) to anticancer drugs. The human major vault protein (MVP) also known as lung resistance-related protein (LRP) represents the predominant component of vaults that may be involved in the defense against xenobiotics. Here, we demonstrate that besides MDR-related cytostatics, also the non-MDR-related drug 5-fluorouracil (5-FU) was able to induce MVP mRNA and protein expression. Treatment with 5-FU amplified the binding activity and interaction of the transcription factor Y-box binding protein-1 (YB-1) with the Y-box of the human MVP gene promoter in a time-dependent manner. 5-FU also induced reporter expressions driven by a panel of newly generated MVP promoter deletion mutants. Interestingly, stably YB-1 overexpressing cell clones showed enhanced binding of YB-1 to the Y-box motif, associated with enhanced basal as well as 5-FU-inducible MVP promoter-driven reporter expressions. Moreover, transduction of YB-1 cDNA led to increased expression of endogenous MVP protein. Under physiological conditions, we observed a strong coexpression of MVP and YB-1 in human colon carcinoma specimen. In summary, our data demonstrate a direct involvement of YB-1 in controlling basal and 5-FU-induced MVP promoter activity. Therefore, YB-1 is directly linked to MVP-mediated drug resistance.

  18. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  19. Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

    Science.gov (United States)

    Saher, Gesine; Quintes, Susanne; Möbius, Wiebke; Wehr, Michael C; Krämer-Albers, Eva-Maria; Brügger, Britta; Nave, Klaus-Armin

    2009-05-13

    Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

  20. Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics.

    Science.gov (United States)

    Mossink, Marieke H; van Zon, Arend; Fränzel-Luiten, Erna; Schoester, Martijn; Kickhoefer, Valerie A; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-12-15

    Vaults are ribonucleoprotein particles with a distinct structure and a high degree of conservation between species. Although no function has been assigned to the complex yet, there is some evidence for a role of vaults in multidrug resistance. To confirm a direct relation between vaults and multidrug resistance, and to investigate other possible functions of vaults, we have generated a major vault protein (MVP/lung resistance-related protein) knockout mouse model. The MVP(-/-) mice are viable, healthy, and show no obvious abnormalities. We investigated the sensitivity of MVP(-/-) embryonic stem cells and bone marrow cells derived from the MVP-deficient mice to various cytostatic agents with different mechanisms of action. Neither the MVP(-/-) embryonic stem cells nor the MVP(-/-) bone marrow cells showed an increased sensitivity to any of the drugs examined, as compared with wild-type cells. Furthermore, the activities of the ABC-transporters P-glycoprotein, multidrug resistance-associated protein and breast cancer resistance protein were unaltered on MVP deletion in these cells. In addition, MVP wild-type and deficient mice were treated with the anthracycline doxorubicin. Both groups of mice responded similarly to the doxorubicin treatment. Our results suggest that MVP/vaults are not directly involved in the resistance to cytostatic agents.

  1. The Human Cytomegalovirus Major Immediate-Early Proteins as Antagonists of Intrinsic and Innate Antiviral Host Responses

    Directory of Open Access Journals (Sweden)

    Michael Nevels

    2009-11-01

    Full Text Available The major immediate-early (IE gene of human cytomegalovirus (CMV is believed to have a decisive role in acute infection and its activity is an important indicator of viral reactivation from latency. Although a variety of gene products are expressed from this region, the 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins are the most abundant and important. Both proteins have long been recognized as promiscuous transcriptional regulators. More recently, a critical role of the IE1 and IE2 proteins in counteracting nonadaptive host cell defense mechanisms has been revealed. In this review we will briefly summarize the available literature on IE1- and IE2-dependent mechanisms contributing to CMV evasion from intrinsic and innate immune responses.

  2. The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Patel, Jital A. [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom); Spisni, Alberto, E-mail: alberto.spisni@unipr.it [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom)

    2009-12-25

    {sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

  3. Effect of rapid rigor mortis processes on protein functionality in pectoralis major muscle of domestic turkeys.

    Science.gov (United States)

    Pietrzak, M; Greaser, M L; Sosnicki, A A

    1997-08-01

    The pale, soft, exudative (PSE) phenomenon in turkey pectoralis major (breast) muscle was studied using a combination of biochemical, meat quality, microscopic, and gel electrophoresis techniques. Breast muscle samples were collected from turkeys characterized by slow vs fast postmortem glycolysis assessed by muscle pH at 20 min after death. The PSE group was characterized by lower muscle ATP (P < .05) and higher lactate levels (P < .05) compared with the normal group. Excess water-holding capacity and cooking yield were significantly lower (P < .05) in the PSE group than in normal turkeys. Breast muscle of the PSE group was also lighter (P < .05) than that in the normal group as determined by Minolta L* values. The SDS-PAGE, Western blotting, and immunofluorescence microscopy revealed that phosphorylase, a soluble enzyme, became tightly associated with the myofibrils in muscle from the PSE group. Also, less myosin could be solubilized from PSE vs normal myofibril samples. The results indicate that irreversible myosin insolubility due to low pH and high-temperature conditions is decisive in the development of PSE turkey breast muscle.

  4. Surface-layer protein A (SlpA is a major contributor to host-cell adherence of Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Michelle M Merrigan

    Full Text Available Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA. In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

  5. Proteome analysis of barley seeds: Identification of major proteins from two-dimensional gels (pl 4-7)

    DEFF Research Database (Denmark)

    Østergaard, O.; Finnie, Christine; Laugesen, S.

    2004-01-01

    inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin......Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water...

  6. Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy.

    Directory of Open Access Journals (Sweden)

    Laurent Dortet

    2011-08-01

    Full Text Available L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP, the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK(- bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.

  7. High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V.; Hurley, Thomas D. (Indiana-Med); (Indiana)

    2010-09-07

    In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.

  8. Genome-wide evolutionary characterization and expression analyses of major latex protein (MLP) family genes in Vitis vinifera.

    Science.gov (United States)

    Zhang, Ningbo; Li, Ruimin; Shen, Wei; Jiao, Shuzhen; Zhang, Junxiang; Xu, Weirong

    2018-04-27

    The major latex protein/ripening-related protein (MLP/RRP) subfamily is known to be involved in a wide range of biological processes of plant development and various stress responses. However, the biological function of MLP/RRP proteins is still far from being clear and identification of them may provide important clues for understanding their roles. Here, we report a genome-wide evolutionary characterization and gene expression analysis of the MLP family in European Vitis species. A total of 14 members, was found in the grape genome, all of which are located on chromosome 1, where are predominantly arranged in tandem clusters. We have noticed, most surprisingly, promoter-sharing by several non-identical but highly similar gene members to a greater extent than expected by chance. Synteny analysis between the grape and Arabidopsis thaliana genomes suggested that 3 grape MLP genes arose before the divergence of the two species. Phylogenetic analysis provided further insights into the evolutionary relationship between the genes, as well as their putative functions, and tissue-specific expression analysis suggested distinct biological roles for different members. Our expression data suggested a couple of candidate genes involved in abiotic stresses and phytohormone responses. The present work provides new insight into the evolution and regulation of Vitis MLP genes, which represent targets for future studies and inclusion in tolerance-related molecular breeding programs.

  9. Effect of white striping myopathy on breast muscle (Pectoralis major) protein turnover and gene expression in broilers.

    Science.gov (United States)

    Vignale, Karen; Caldas, Justina V; England, Judy A; Boonsinchai, Nirun; Magnuson, Andrew; Pollock, Erik D; Dridi, Sami; Owens, Casey M; Coon, Craig N

    2017-04-01

    A study was conducted to evaluate the effect of white striping ( ) of broiler breast muscle ( Pectoralis major ) on protein turnover and gene expression of genes related to protein degradation and fatty acid synthesis. A total of 560 day-old male broiler chicks Cobb 500 were allocated in a total of 16 pens, 35 chicks per pen. A completely randomized design was conducted with a 2 × 3 factorial arrangement (2 scores: severe and normal, and 3 breast meat samples sites). At d 60, 20 birds were randomly selected, euthanized, and scored for white striping. Scoring was either normal ( , no WS) or severe ( ). Also, the same day, 17 birds (16 infused, one control) were randomly selected and infused with a solution of 15 N Phen 40% ( ). Breast muscle tissue was taken for gene expression analysis of the following genes: MuRF1, atrogin-1, IGF-1, insulin receptor ( ), fatty acid synthetase, and acetyl CoA carboxylase ( ). Each bird was humanely euthanized after 10 minutes of infusion and scored for WS (NORM or SEV). Samples of the breast muscle ( Pectoralis major ) were taken at different layers (3 samples per bird: ventral, medial, dorsal), along with a sample of excreta for 3-methylhistidine analysis. Out of the 16 breast samples taken, only 10 were selected for analysis based on the WS score (5 NORM and 5 SEV). No significant differences ( P > 0.05) were found in fractional synthesis rate ( ) between SEV WS, NORM and sample sites for breast meat. However, fractional breakdown rate ( ) was significantly higher in birds with SEV WS compared to NORM (8.2 and 4.28, respectively, P white striping are degrading more muscular protein and mobilizing more fat. © 2016 Poultry Science Association Inc.

  10. A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

    Science.gov (United States)

    Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

    2016-03-25

    Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

  12. Association of translocator protein total distribution volume with duration of untreated major depressive disorder: a cross-sectional study.

    Science.gov (United States)

    Setiawan, Elaine; Attwells, Sophia; Wilson, Alan A; Mizrahi, Romina; Rusjan, Pablo M; Miler, Laura; Xu, Cynthia; Sharma, Sarita; Kish, Stephen; Houle, Sylvain; Meyer, Jeffrey H

    2018-04-01

    People with major depressive disorder frequently exhibit increasing persistence of major depressive episodes. However, evidence for neuroprogression (ie, increasing brain pathology with longer duration of illness) is scarce. Microglial activation, which is an important component of neuroinflammation, is implicated in neuroprogression. We examined the relationship of translocator protein (TSPO) total distribution volume (V T ), a marker of microglial activation, with duration of untreated major depressive disorder, and with total illness duration and antidepressant exposure. In this cross-sectional study, we recruited participants aged 18-75 years from the Toronto area and the Centre for Addiction and Mental Health (Toronto, ON, Canada). Participants either had major depressive episodes secondary to major depressive disorder or were healthy, as confirmed with a structured clinical interview and consultation with a study psychiatrist. To be enrolled, participants with major depressive episodes had to score a minimum of 17 on the 17-item Hamilton Depression Rating Scale, and had to be medication free or taking a stable dose of medication for at least 4 weeks before PET scanning. Eligible participants were non-smokers; had no history of or concurrent alcohol or substance dependence, neurological illness, autoimmune disorder, or severe medical problems; and were free from acute medical illnesses for the previous 2 weeks before PET scanning. Participants were excluded if they had used brain stimulation treatments within the 6 months before scanning, had used anti-inflammatory drugs lasting at least 1 week within the past month, were taking hormone replacement therapy, had psychotic symptoms, had bipolar disorder (type I or II) or borderline antisocial personality disorder, or were pregnant or breastfeeding. We scanned three primary grey-matter regions of interest (prefrontal cortex, anterior cingulate cortex, and insula) and 12 additional regions and subregions using 18

  13. Tumor Epression of Major Vault Protein is an Adverse Prognostic Factor for Radiotherapy Outcome in Oropharyngeal Carcinoma

    International Nuclear Information System (INIS)

    Silva, Priyamal; West, Catharine M.; Slevin, Nick F.R.C.R.; Valentine, Helen; Ryder, W. David J. Grad. I.S.; Hampson, Lynne; Bibi, Rufzan; Sloan, Philip; Thakker, Nalin; Homer, Jarrod; Hampson, Ian

    2007-01-01

    Purpose: Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport, with the major vault protein (MVP or lung resistance-related protein [LRP]) being the main component. The MVP gene is located on chromosome 16 close to the multidrug resistance-associated protein and protein kinase c-β genes. The role of MVP in cancer drug resistance has been demonstrated in various cell lines as well as in ovarian carcinomas and acute myeloid leukemia, but nothing is known about its possible role in radiation resistance. Our aim was to examine this in head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: Archived biopsy material was obtained for 78 patients with squamous cell carcinoma of the oropharynx who received primary radiotherapy with curative intent. Immunohistochemistry was used to detect MVP expression. Locoregional failure and cancer-specific survival were estimated using cumulative incidence and Cox multivariate analyses. Results: In a univariate and multivariate analysis, MVP expression was strongly associated with both locoregional failure and cancer-specific survival. After adjustment for disease site, stage, grade, anemia, smoking, alcohol, gender, and age, the estimated hazard ratio for high MVP (2/3) compared with low (0/1) was 4.98 (95% confidence interval, 2.17-11.42; p 0.0002) for locoregional failure and 4.28 (95% confidence interval, 1.85-9.95; p = 0.001) for cancer-specific mortality. Conclusion: These data are the first to show that MVP may be a useful prognostic marker associated with radiotherapy resistance in a subgroup of patients with HNSCC

  14. Predictive Value of C-Reactive Protein for Major Complications after Major Abdominal Surgery: A Systematic Review and Pooled-Analysis.

    Directory of Open Access Journals (Sweden)

    Jennifer Straatman

    Full Text Available Early diagnosis and treatment of complications after major abdominal surgery can decrease associated morbidity and mortality. Postoperative CRP levels have shown a strong correlation with complications. Aim of this systematic review and pooled-analysis was to assess postoperative values of CRP as a marker for major complications and construct a prediction model.A systematic review was performed for CRP levels as a predictor for complications after major abdominal surgery (MAS. Raw data was obtained from seven studies, including 1427 patients. A logit regression model assessed the probability of major complications as a function of CRP levels on the third postoperative day. Two practical cut-offs are proposed: an optimal cut-off for safe discharge in a fast track protocol and another for early identification of patients with increased risk for major complications.A prediction model was calculated for major complications as a function of CRP levels on the third postoperative day. Based on the model several cut-offs for CRP are proposed. For instance, a two cut-off system may be applied, consisting of a safe discharge criterion with CRP levels below 75 mg/L, with a negative predictive value of 97.2%. A second cut-off is set at 215 mg/L (probability 20% and serves as a predictor of complications, indicating additional CT-scan imaging.The present study provides insight in the interpretation of CRP levels after major abdominal surgery, proposing a prediction model for major complications as a function of CRP on postoperative day 3. Cut-offs for CRP may be implemented for safe early-discharge in a fast-track protocol and, secondly as a threshold for additional examinations, such as CT-scan imaging, even in absence of clinical signs, to confirm or exclude major complications. The prediction model allows for setting a cut-off at the discretion of individual surgeons or surgical departments.

  15. Biological assessment of neonicotinoids imidacloprid and its major metabolites for potentially human health using globular proteins as a model.

    Science.gov (United States)

    Ding, Fei; Peng, Wei

    2015-06-01

    The assessment of biological activities of imidacloprid and its two major metabolites, namely 6-chloronicotinic acid and 2-imidazolidone for nontarget organism, by employing essentially functional biomacromolecules, albumin and hemoglobin as a potentially model with the use of circular dichroism (CD), fluorescence, extrinsic 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence as well as molecular modeling is the theme of this work. By dint of CD spectra and synchronous fluorescence, it was clear that the orderly weak interactions between amino acid residues within globular proteins were disturbed by imidacloprid, and this event led to marginally alterations or self-regulations of protein conformation so as to lodge imidacloprid more tightly. Both steady state and time-resolved fluorescence suggested that the fluorescence of Trp residues in proteins was quenched after the presence of imidacloprid, corresponding to noncovalent protein-imidacloprid complexes formation and, the reaction belongs to moderate association (K=1.888/1.614×10(4)M(-1) for albumin/hemoglobin-imidacloprid, respectively), hydrogen bonds and π stacking performed a vital role in stabilizing the complexes, as derived from thermodynamic analysis and molecular modeling. With the aid of hydrophobic ANS experiments, subdomain IIA and α1β2 interface of albumin and hemoglobin, respectively, were found to be preserved high-affinity for imidacloprid. These results ties in with the subsequently molecular modeling laying imidacloprid in the Sudlow's site I and close to Trp-213 residue on albumin, while settling down B/Trp-37 residue nearby in hemoglobin, and these conclusions further confirmed by site-directed mutagenesis and molecular dynamics simulation. But, at the same time, several crucial noncovalent bonds came from other amino acid residues, e.g. Arg-194 and Arg-198 (albumin) and B/Arg-40, B/Asp-99 and B/Asn-102 (hemoglobin) cannot be ignored completely. Based on the comparative studies of

  16. Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

    Science.gov (United States)

    Griffith, I J; Smith, P M; Pollock, J; Theerakulpisut, P; Avjioglu, A; Davies, S; Hough, T; Singh, M B; Simpson, R J; Ward, L D

    1991-02-25

    We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

  17. Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

    Science.gov (United States)

    Ventas, P; Carreira, J; Polo, F

    1991-08-01

    The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

  18. Biotic and environmental stress induces nitration and changes in structure and function of the sea urchin major yolk protein toposome.

    Science.gov (United States)

    Castellano, Immacolata; Migliaccio, Oriana; Ferraro, Giarita; Maffioli, Elisa; Marasco, Daniela; Merlino, Antonello; Zingone, Adriana; Tedeschi, Gabriella; Palumbo, Anna

    2018-03-15

    The major yolk protein toposome plays crucial roles during gametogenesis and development of sea urchins. We previously found that nitration of toposome increases in the gonads of a Paracentrotus lividus population living in a marine protected area affected by toxic blooms of Ostreospsis cf. ovata, compared to control populations. This modification is associated with ovatoxin accumulation, high levels of nitric oxide in the gonads, and a remarkable impairment of progeny development. However, nothing is known about the environmental-mediated-regulation of the structure and biological function of toposome. Here, we characterize through wide-ranging biochemical and structural analyses the nitrated toposome of sea urchins exposed to the bloom, and subsequently detoxified. The increased number of nitrated tyrosines in toposome of sea urchins collected during algal bloom induced structural changes and improvement of the Ca 2+ -binding affinity of the protein. After 3 months' detoxification, ovatoxin was undetectable, and the number of nitric oxide-modified tyrosines was reduced. However, the nitration of specific residues was irreversible and occurred also in embryos treated with metals, used as a proxy of environmental pollutants. The structural and functional changes of toposome caused by nitration under adverse environmental conditions may be related to the defective development of sea urchins' progeny.

  19. Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

    International Nuclear Information System (INIS)

    Zheng Chunfu; Brownlie, Robert; Babiuk, Lorne A.; Hurk, Sylvia van Drunen Littel-van den

    2004-01-01

    Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ( 51 RRPR 54 ) or pat7 ( 48 PRVRRPR 54 ) NLS2 abrogated nuclear accumulation, whereas deletion of 48 PRV 50 did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ( 11 RRPRR 15 ), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids 485 LSAYLTLFVAL 495 . This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm

  20. Progranulin, a major secreted protein of mouse adipose-derived stem cells, inhibits light-induced retinal degeneration.

    Science.gov (United States)

    Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

    2014-01-01

    Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

  1. The Major Chromophore Arising from Glucose Degradation and Oxidative Stress Occurrence during Lens Proteins Glycation Induced by Glucose

    Directory of Open Access Journals (Sweden)

    Felipe Ávila

    2017-12-01

    Full Text Available Glucose autoxidation has been proposed as a key reaction associated with deleterious effects induced by hyperglycemia in the eye lens. Little is known about chromophores generated during glucose autoxidation. In this study, we analyzed the effect of oxidative and dicarbonyl stress in the generation of a major chromophore arising from glucose degradation (GDC and its association with oxidative damage in lens proteins. Glucose (5 mM was incubated with H2O2 (0.5–5 mM, Cu2+ (5–50 μM, glyoxal (0.5–5 mM or methylglyoxal (0.5–5 mM at pH 7.4, 5% O2, 37 °C, from 0 to 30 days. GDC concentration increased with incubation time, as well as when incubated in the presence of H2O2 and/or Cu2+, which were effective even at the lowest concentrations. Dicarbonylic compounds did not increase the levels of GDC during incubations. 1H, 13C and FT-IR spectra from the purified fraction containing the chromophore (detected by UV/vis spectroscopy showed oxidation products of glucose, including gluconic acid. Lens proteins solutions (10 mg/mL incubated with glucose (30 mM presented increased levels of carboxymethyl-lysine and hydrogen peroxide that were associated with GDC increase. Our results suggest a possible use of GDC as a marker of autoxidative reactions occurring during lens proteins glycation induced by glucose.

  2. Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA- or Virus-Induced Proinflammatory Response.

    Science.gov (United States)

    Peng, Nanfang; Liu, Shi; Xia, Zhangchuan; Ren, Sheng; Feng, Jian; Jing, Mingzhen; Gao, Xin; Wiemer, Erik A C; Zhu, Ying

    2016-03-15

    Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP(-/-)) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)β-liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPβ binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPβ. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP(-/-) mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J

    1988-01-01

    markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those...... of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition...

  4. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  5. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Science.gov (United States)

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  6. Diagnostic value of C-reactive protein to rule out infectious complications after major abdominal surgery: a systematic review and meta-analysis

    NARCIS (Netherlands)

    Gans, Sarah L.; Atema, Jasper J.; van Dieren, Susan; Groot Koerkamp, Bas; Boermeester, Marja A.

    2015-01-01

    Infectious complications occur frequently after major abdominal surgery and have a major influence on patient outcome and hospital costs. A marker that can rule out postoperative infectious complications (PICs) could aid patient selection for safe and early hospital discharge. C-reactive protein

  7. Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

    Science.gov (United States)

    Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-01-01

    Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

  8. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

    Science.gov (United States)

    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  9. Elevated Hippocampal Cholinergic Neurostimulating Peptide precursor protein (HCNP-pp) mRNA in the amygdala in major depression.

    Science.gov (United States)

    Bassi, Sabrina; Seney, Marianne L; Argibay, Pablo; Sibille, Etienne

    2015-04-01

    The amygdala is innervated by the cholinergic system and is involved in major depressive disorder (MDD). Evidence suggests a hyper-activate cholinergic system in MDD. Hippocampal Cholinergic Neurostimulating Peptide (HCNP) regulates acetylcholine synthesis. The aim of the present work was to investigate expression levels of HCNP-precursor protein (HCNP-pp) mRNA and other cholinergic-related genes in the postmortem amygdala of MDD patients and matched controls (females: N = 16 pairs; males: N = 12 pairs), and in the mouse unpredictable chronic mild stress (UCMS) model that induced elevated anxiety-/depressive-like behaviors (females: N = 6 pairs; males: N = 6 pairs). Results indicate an up-regulation of HCNP-pp mRNA in the amygdala of women with MDD (p < 0.0001), but not males, and of UCMS-exposed mice (males and females; p = 0.037). HCNP-pp protein levels were investigated in the human female cohort, but no difference was found. There were no differences in gene expression of acetylcholinesterase (AChE), muscarinic (mAChRs) or nicotinic receptors (nAChRs) between MDD subjects and controls or UCMS and control mice, except for an up-regulation of AChE in UCMS-exposed mice (males and females; p = 0.044). Exploratory analyses revealed a baseline expression difference of cholinergic signaling-related genes between women and men (p < 0.0001). In conclusion, elevated amygdala HCNP-pp expression may contribute to mechanisms of MDD in women, potentially independently from regulating the cholinergic system. The differential expression of genes between women and men could also contribute to the increased vulnerability of females to develop MDD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Further Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with ICP8, the Major DNA-Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1994-01-01

    Baringer, J.R. 1974. Recovery of herpes simplex virus from human sacral ganglions. N. Eng!. J. Med. 291:828-830. Baringer, J.R. 1976. The biology of herpes ...UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA~Binding Protein of Herpes Simplex Virus" beyond brief...Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA-Binding Protein of Herpes Simplex Virus Allen G. Albright Doctor of

  11. Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses.

    Science.gov (United States)

    Rowe, Janet M; Fabre, Marie-Françoise; Gobena, Daniel; Wilson, William H; Wilhelm, Steven W

    2011-05-01

    Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene. © 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

  12. Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality.

    Science.gov (United States)

    Xing, T; Wang, M F; Han, M Y; Zhu, X S; Xu, X L; Zhou, G H

    2017-09-01

    Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH24 h values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (Pmeat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.

  13. Amyloid β Is Not the Major Factor Accounting for Impaired Adult Hippocampal Neurogenesis in Mice Overexpressing Amyloid Precursor Protein

    Directory of Open Access Journals (Sweden)

    Hongyu Pan

    2016-10-01

    Full Text Available Adult hippocampal neurogenesis was impaired in several Alzheimer's disease models overexpressing mutant human amyloid precursor protein (hAPP. However, the effects of wild-type hAPP on adult neurogenesis and whether the impaired adult hippocampal neurogenesis was caused by amyloid β (Aβ or APP remained unclear. Here, we found that neurogenesis was impaired in the dentate gyrus (DG of adult mice overexpressing wild-type hAPP (hAPP-I5 compared with controls. However, the adult hippocampal neurogenesis was more severely impaired in hAPP-I5 than that in hAPP-J20 mice, which express similar levels of hAPP mRNA but much higher levels of Aβ. Furthermore, reducing Aβ levels did not affect the number of doublecortin-positive cells in the DG of hAPP-J20 mice. Our results suggested that hAPP was more likely an important factor inhibiting adult neurogenesis, and Aβ was not the major factor affecting neurogenesis in the adult hippocampus of hAPP mice.

  14. Absence of association between major vault protein (MVP) gene polymorphisms and drug resistance in Chinese Han patients with partial epilepsy.

    Science.gov (United States)

    Zhou, Luo; Zhang, Mengqi; Long, Hongyu; Long, Lili; Xie, Yuanyuan; Liu, Zhaoqian; Kang, Jin; Chen, Qihua; Feng, Li; Xiao, Bo

    2015-11-15

    Drug resistance in epilepsy is common despite many antiepileptic drugs (AEDs) available for treatment. The development of drug resistant epilepsy may be a result of multiple factors. Several previous studies reported that the major vault protein (MVP) was significantly increased in epileptogenic brain tissues resected from patients with partial-onset seizures, indicating the possible involvement of MVP in drug resistance. In this article, we aimed to identify the association between single nucleotide polymorphisms (SNPs) of MVP gene and drug resistance of partial epilepsy in a Chinese Han population. A total of 510 patients with partial-onset seizures and 206 healthy controls were recruited. Among the patients, 222 were drug resistant and 288 were responsive. The selection of tagging SNPs was based on the Hapmap database and Haploview software and the genotyping was conducted on the Sequenom MassARRAY iPLEX platform. For the selected loci rs12149746, rs9938630 and rs4788186 in the MVP gene, there was no significant difference in allele or genotype distribution between the drug resistant and responsive groups, or between all of the patients and healthy controls. Linkage disequilibrium between any two loci was detected but there was no significant difference in haplotype frequency between the drug resistant and responsive groups. Our results suggest that MVP genetic polymorphisms and haplotypes may not be associated with drug resistance of partial epilepsy in the Chinese Han population. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Major vault protein (MVP) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis.

    Science.gov (United States)

    Balan, Shabeesh; Radhab, Saradalekshmi Koramannil; Radha, Koramannil; Sathyan, Sanish; Vijai, Joseph; Banerjee, Moinak; Radhakrishnan, Kurupath

    2013-09-10

    The human major vault protein (MVP) has been implicated in the development of drug resistance in cancer cells. Over expression of MVP has also been reported in brain tissue samples from antiepileptic drug (AED)-resistant human focal epilepsies. To investigate the relationship between single nucleotide polymorphisms (SNPs) involving the MVP gene and AED-resistance, we compared the distribution of three SNPs in the MVP gene, rs4788187, rs3815824 and rs3815823, among 220 patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), 201 patients with juvenile myoclonic epilepsy (JME) (prototype of AED-responsive epilepsy syndrome) and 213 ethnically matched non-epilepsy controls. All the patients and controls were residents of the South Indian state of Kerala for more than three generations. We did not find any significant difference in allele and genotypic frequencies of the studied SNPs between AED-resistant and AED-responsive cohorts, and between AED-resistant and AED-responsive cohorts independently and pooled together when compared with the controls. We conclude that rs4788187, rs3815824, rs3815823 variants of the MVP gene are associated neither with predisposition for epilepsy nor with AED-resistance in the population that we have studied. Our results suggest the need for further research into the link between MVP and AED-resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages*

    Science.gov (United States)

    Ben, Jingjing; Zhang, Yan; Zhou, Rongmei; Zhang, Haiyang; Zhu, Xudong; Li, Xiaoyu; Zhang, Hanwen; Li, Nan; Zhou, Xiaodan; Bai, Hui; Yang, Qing; Li, Donghai; Xu, Yong; Chen, Qi

    2013-01-01

    Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis. PMID:23703615

  17. Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.).

    Science.gov (United States)

    Conlan, R S; Griffiths, L A; Napier, J A; Shewry, P R; Mantell, S; Ainsworth, C

    1995-06-01

    cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

  18. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    Energy Technology Data Exchange (ETDEWEB)

    Dhondt, Ineke; Petyuk, Vladislav A.; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D.; Depuydt, Geert; Braeckman, Bart P.

    2016-09-01

    Cellular protein quality can be maintained by proteolytic elimination of damaged proteins and replacing them with newly synthesized copies, a process called protein turnover (Ward, 2000). Protein turnover rates have been estimated using SILAC (stable isotope labeling by amino acids in cell culture) in prokaryotes and eukaryotes. The last decade has witnessed a growing interest in the analysis of whole-organism proteome dynamics in metazoans using the same approach (Claydon and Beynon, 2012). In recent work, SILAC was applied to monitor protein synthesis throughout life in adult Caenorhabditis elegans (Vukoti et al., 2015) and to investigate food intake (Gomez-Amaro et al., 2015

  19. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of

  20. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

    Science.gov (United States)

    Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

    2006-01-01

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

  1. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    International Nuclear Information System (INIS)

    Prasad, C. Krishna; Meyers, Craig; Zhan Dejin; You Hong; Chiriva-Internati, Maurizio; Mehta, Jawahar L.; Liu Yong; Hermonat, Paul L.

    2003-01-01

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

  2. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  3. Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

    DEFF Research Database (Denmark)

    Ostergaard, P; Phan, H; Johansen, L B

    1998-01-01

    The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one prim...... approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex....

  4. Identification of a major non-structural protein in the nuclei of Rift Valley fever virus-infected cells.

    Science.gov (United States)

    Struthers, J K; Swanepoel, R

    1982-06-01

    A non-structural protein of mol. wt. 34 X 10(3) was demonstrated in the nuclei of Rift Valley fever virus-infected Vero cells by SDS-polyacrylamide gel electro-phoresis. The protein appears to correspond to the virus-induced antigen demonstrated by indirect immunofluorescence in intranuclear inclusions.

  5. Phylogenetic analysis of members of the Phycodnaviridae virus family, using amplified fragments of the major capsid protein gene.

    Science.gov (United States)

    Larsen, J B; Larsen, A; Bratbak, G; Sandaa, R-A

    2008-05-01

    Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family Phycodnaviridae are also interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the prymnesiophyte Pyramimonas orientalis and the haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis virus 01B [PoV-01B], Phaeocystis pouchetii virus 01 [PpV-01], and Chrysochromulina ericina virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the Phycodnaviridae family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this

  6. Anal lymphogranuloma venereum infection screening with IgA anti-Chlamydia trachomatis-specific major outer membrane protein serology.

    Science.gov (United States)

    de Vries, Henry J C; Smelov, Vitaly; Ouburg, Sander; Pleijster, Jolein; Geskus, Ronald B; Speksnijder, Arjen G C L; Fennema, Johannes S A; Morré, Servaas A

    2010-12-01

    Anal lymphogranuloma venereum (LGV) infections, caused by Chlamydia trachomatis biovar L (Ct+/LGV+), are endemic among men who have sex with men (MSM). Anal non-LGV biovar Ct infections (Ct+/LGV-) can be eradicated with 1 week doxycycline, whereas Ct+/LGV+ infections require 3-week doxycycline. To differentiate Ct+/LGV+ from Ct+/LGV- infections, biovar-specific Nucleic Acid Amplification Test (NAAT) are standard, but also expensive and laborious. A chlamydia-specific serological assay could serve as an alternative test. MSM were screened for anal Ct+/LGV+ and Ct+/LGV- infections with a commercial nonspecific NAAT and an in house biovar L-specific NAAT. Serum samples were evaluated with chlamydia-specific anti-Major Outer Membrane Protein (MOMP) and antilipopolysaccharide assays of IgA and IgG classes. Asymptomatic patients were identified as: (1) no anal complaints or (2) no microscopic inflammation (i.e., <10 leucocytes per high power field in anal smears). The best differentiating assay was subsequently evaluated in 100 Ct+/LGV+ and 100 Ct+/LGV- MSM using different cut-off points. The anti-MOMP IgA assay was the most accurate to differentiate Ct+/LGV+ (n = 42) from Ct+/LGV- (n = 19) with 85.7% sensitivity (95% confidence interval [CI], 72.2-93.3) and 84.2% specificity (95% CI, 62.4-94.5), even among asymptomatic patients. In a population comprising 98 Ct+/LGV+ and 105 Ct+/LGV- patients, the anti-MOMP IgA assay scored most accurate when the cut-off point was set to 2.0 with 75.5% (95% CI, 65.8-83.6) sensitivity and 74.3% (95% CI, 64.8-82.3) specificity. The IgA anti-MOMP assay can identify a considerable proportion of the (asymptomatic) anal LGV infections correctly. Yet, biovar L-specific NAAT are still the preferred diagnostic tests in clinical settings.

  7. FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein

    NARCIS (Netherlands)

    Nazarov, P.V.; Koehorst, R.B.M.; Vos, W.L.; Apanasovich, V.V.; Hemminga, M.A.

    2007-01-01

    A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based

  8. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    Science.gov (United States)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  9. Expression and cellular distribution of major vault protein: a putative marker for pharmacoresistance in a rat model for temporal lobe epilepsy

    NARCIS (Netherlands)

    van Vliet, Erwin A.; Aronica, Eleonora; Redeker, Sandra; Gorter, Jan A.

    2004-01-01

    PURPOSE: Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy. METHODS: By using real-time polymerase chain reaction (PCR)

  10. Expression and Cellular Distribution of Major Vault Protein: A Putative Marker for Pharmacoresistance in a Rat Model for Temporal Lobe Epilepsy

    NARCIS (Netherlands)

    Vliet van, E.A.; Aronica, E.; Redeker, S.; Gorter, J.A.

    2004-01-01

    Summary: Purpose: Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy. Methods: By using real-time polymerase chain

  11. Coagulopathy following major liver resection: the effect of rBPI21 and the role of decreased synthesis of regulating proteins by the liver

    NARCIS (Netherlands)

    Meijer, C.; Wiezer, M. J.; Hack, C. E.; Boelens, P. G.; Wedel, N. I.; Meijer, S.; Nijveldt, R. J.; Statius Muller, M. G.; Wiggers, T.; Zoetmulder, F. A.; Borel Rinkes, I. H.; Cuesta, M. A.; Gouma, D. J.; van de Velde, C. J.; Tilanus, H. W.; Scotté, M.; Thijs, L. G.; van Leeuwen, P. A.

    2001-01-01

    This prospective study investigated the role of reduced hepatic synthesis of regulating proteins in coagulopathy after partial hepatectomy (PH) compared with major abdominal surgery (MAS) without involvement of the liver. Furthermore, we studied the effect of rBPI21, an endotoxin-neutralizing agent,

  12. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    International Nuclear Information System (INIS)

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-01-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

  13. Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

    OpenAIRE

    Kang, Seung-Won; Kweon, Chang-Hee; Choi, Eun-Jin; Yoon, Yong-Dhuk

    1999-01-01

    To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein ...

  14. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    International Nuclear Information System (INIS)

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and 125 I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25 0 C instead of 100 0 C

  15. Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1992-10-20

    R . 1974 . Recovery of herpes simplex virus from human sacral gangl ions. N. Engl. J. Med. 291 :828-830. Baringer, J.R . 1975. Herpes simplex virus...AII’I fORCE MEDICAL C(NTEIt Title of Dissertation : "Ideatification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and...Demonstration that It Interacts with reps. the Major DNA Binding Protein of Herpes Simplex Virus" Name of Candidate: Lisa Shelton Doctor of

  16. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  17. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  18. Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

    Science.gov (United States)

    Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

    2016-08-01

    Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. The Mr 30,000-33,000 major protein components of the lateral elements of synaptonemal complexes of the rat

    NARCIS (Netherlands)

    Lammers, H.

    1999-01-01

    Synaptonemal complexes (SCs) are intranuclear structures which are formed during meiotic prophase between homologous chromosomes. The SC consists of two protein-rich axes, either of which is found at the basis of one of the homologous chromosomes. These axes, called lateral elements (LEs),

  20. Evolution of species-specific major seminal fluid proteins in placental mammals by gene death and positive selection

    NARCIS (Netherlands)

    Meslin, C.; Laurin, M.; Callebaut, I.; Druart, X.; Monget, P.

    2015-01-01

    The seminal fluid is a complex substance composed of a variety of secreted proteins and has been shown to play an important role in the fertilisation process in mammals and also in Drosophila. Several genes under positive selection have been documented in some rodents and primates. Our study

  1. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    Science.gov (United States)

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  2. Surfactant protein D augments bacterial association but attenuates major histocompatibility complex class II presentation of bacterial antigens

    DEFF Research Database (Denmark)

    Hansen, Søren; Lo, Bernice; Evans, Kathy

    2006-01-01

    Development of dementia, including Alzheimer's disease (AD), is associated with lipid dysregulation and inflammation. As the host defense lectin surfactant protein D (SP-D) has multiple effects in lipid homeostasis and inflammation, the correlation between SP-D concentrations and development of d...

  3. Co-suppression of synthesis of major x-kafirin sub-class together with y-kafirin-1 and y-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum

    CSIR Research Space (South Africa)

    Grootboom, AW

    2014-01-01

    Full Text Available Co-suppressing major kafirin sub-classes is fundamental to improved protein digestibility and nutritional value of sorghum. The improvement is linked to an irregularly invaginated phenotype of protein bodies....

  4. The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

    International Nuclear Information System (INIS)

    Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo

    2005-01-01

    The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F WT ) and attachment (H WT ) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H WT determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F WT reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection

  5. Dioscorin, the major tuber storage protein of yam (Dioscorea batatas decne) with carbonic anhydrase and trypsin inhibitor activities.

    Science.gov (United States)

    Hou, W C; Liu, J S; Chen, H J; Chen, T E; Chang, C F; Lin, Y H

    1999-05-01

    Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

  6. Alternate phase variation in expression of two major surface membrane proteins (MBA and UU376) of Ureaplasma parvum serovar 3.

    Science.gov (United States)

    Zimmerman, Carl-Ulrich R; Stiedl, Thomas; Rosengarten, Renate; Spergser, Joachim

    2009-03-01

    Ureaplasma urealyticum and Ureaplasma parvum are commensals and pathogens of the human urogenital tract and of newborn infants. There are four distinct U. parvum serovars and 10 distinct U. urealyticum serovars. Both species possess a distinct immunodominant variable surface protein, the multiple banded antigen (MBA), which shows size variability among isolates as a result of changes in the number of C-terminal repeating units. Adjacent to the MBA gene (UU375) lies UU376, which was annotated as 'Ureaplasma-specific conserved hypothetical gene'. In four different strains of U. parvum serovar 3, we demonstrated expression of UU376 by Western blot analysis and phase variation between UU376, here designated Upvmp376 (Ureaplasma phase-variable membrane protein 376), and MBA after application of selective pressure with hyperimmune antisera directed against either protein. By Southern blot analysis, we found that the switch between MBA and Upvmp376 expression is associated with a DNA inversion event in which the nonrepetitive region of the MBA gene and its putative promoter region are opposed to either the repetitive region of MBA or UU376. We propose that in U. parvum serovar 3, and presumably in all U. parvum and U. urealyticum, an inversion event at specific sites effects an alternate ON/OFF switching of the genes UU375 and UU376.

  7. Ribosomal dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in Lactococcus lactis.

    Science.gov (United States)

    Breüner, Anne; Frees, Dorte; Varmanen, Pekka; Boguta, Anna Monika; Hammer, Karin; Martinussen, Jan; Kilstrup, Mogens

    2016-10-01

    We analysed the response of the model bacterium Lactococcus lactis to abrupt depletion of glucose after several generations of exponential growth. Glucose depletion resulted in a drastic drop in the energy charge accompanied by an extremely low GTP level and an almost total arrest of protein synthesis. Strikingly, the cell prioritized the continued synthesis of a few proteins, of which the ribosomal dimerization factor YfiA was the most highly expressed. Transcriptome analysis showed no immediate decrease in total mRNA levels despite the lowered nucleotide pools and only marginally increased levels of the yfiA transcript. Severe up-regulation of genes in the FruR, CcpA, ArgR and AhrC regulons were consistent with a downshift in carbon and energy source. Based upon the results, we suggest that transcription proceeded long enough to record the transcriptome changes from activation of the FruR, CcpA, ArgR and AhrC regulons, while protein synthesis stopped due to an extremely low GTP concentration emerging a few minutes after glucose depletion. The yfiA deletion mutant exhibited a longer lag phase upon replenishment of glucose and a faster death rate after prolonged starvation supporting that YfiA-mediated ribosomal dimerization is important for keeping long-term starved cells viable and competent for growth initiation.

  8. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    Science.gov (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  9. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants

    OpenAIRE

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri...

  10. Recombinant major urinary proteins of the mouse in specific IgE and IgG testing

    NARCIS (Netherlands)

    Krop, Esmeralda J. M.; Matsui, Elizabeth C.; Sharrow, Scott D.; Stone, Martin J.; Gerber, Peter; van der Zee, Jaring S.; Chapman, Martin D.; Aalberse, Rob C.

    2007-01-01

    BACKGROUND: Recombinant allergens are preferred over natural allergen extracts in measuring antibodies. We tested the use of recombinant variants of the major mouse allergen Mus m 1 in detection of mouse-specific antibodies in sera of laboratory animal workers and children. METHODS: Six recombinant

  11. The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

    Science.gov (United States)

    Sean, Polen; Nguyen, Joseph H C; Semler, Bert L

    2008-09-01

    Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

  12. Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

    Science.gov (United States)

    Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

    2014-09-01

    Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

  13. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis

    DEFF Research Database (Denmark)

    Palmer, Colin N A; Irvine, Alan D; Terron-Kwiatkowski, Ana

    2006-01-01

    most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic...... variants (R510X and 2282del4) in the gene encoding filaggrin (FLG) are very strong predisposing factors for atopic dermatitis. These variants are carried by approximately 9% of people of European origin. These variants also show highly significant association with asthma occurring in the context of atopic...

  14. The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris.

    Science.gov (United States)

    Kolarich, Daniel; Léonard, Renaud; Hemmer, Wolfgang; Altmann, Friedrich

    2005-10-01

    Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.

  15. Anaplasma marginale major surface protein 1a: a marker of strain diversity with implications for control of bovine anaplasmosis.

    Science.gov (United States)

    Cabezas-Cruz, Alejandro; de la Fuente, José

    2015-04-01

    Classification of bacteria is challenging due to the lack of a theory-based framework. In addition, the adaptation of bacteria to ecological niches often results in selection of strains with diverse virulence, pathogenicity and transmission characteristics. Bacterial strain diversity presents challenges for taxonomic classification, which in turn impacts the ability to develop accurate diagnostics and effective vaccines. Over the past decade, the worldwide diversity of Anaplasma marginale, an economically important tick-borne pathogen of cattle, has become apparent. The extent of A. marginale strain diversity, formerly underappreciated, has contributed to the challenges of classification which, in turn, likely impacts the design and development of improved vaccines. Notably, the A. marginale surface protein 1a (MSP1a) is a model molecule for these studies because it serves as a marker for strain identity, is both an adhesin necessary for infection of cells and an immuno-reactive protein and is also an indicator of the evolution of strain diversity. Herein, we discuss a molecular taxonomic approach for classification of A. marginale strain diversity. Taxonomic analysis of this important molecule provides the opportunity to understand A. marginale strain diversity as it relates geographic and ecological factors and to the development of effective vaccines for control of bovine anaplasmosis worldwide. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair.

    Science.gov (United States)

    Kato-Inui, Tomoko; Takahashi, Gou; Hsu, Szuyin; Miyaoka, Yuichiro

    2018-05-18

    Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.

  17. Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex

    DEFF Research Database (Denmark)

    Sørensen, Charlotte B; Andresen, Brage S; Jensen, Uffe B

    2003-01-01

    vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection...... of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated...... with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P

  18. Mutagens from the cooking of food. II. Survey by Ames/Salmonella test of mutagen formation in the major protein-rich foods of the American diet

    Energy Technology Data Exchange (ETDEWEB)

    Bjeldanes, L.F. (Univ. of California, Berkeley); Morris, M.M.; Felton, J.S.; Healy, S.; Stuermer, D.; Berry, P.; Timourian, H.; Hatch, F.T.

    1982-01-01

    The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA--ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon--were examined for their mutagenicity towards S. typhimurium TA1538 after normal 'household' cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling at 100-475/sup 0/C). Well-done fried ground beef, beef steak, ham, pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs andd egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperature above 225/sup 0/C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.

  19. Mutagens from the cooking of food. II. Survey by Ames/Salmonella test of mutagen formation in the major protein-rich foods of the American diet.

    Science.gov (United States)

    Bjeldanes, L F; Morris, M M; Felton, J S; Healy, S; Stuermer, D; Berry, P; Timourian, H; Hatch, F T

    1982-08-01

    The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA--ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon--were examined for their mutagenicity towards S. typhimurium TA1538 after normal 'household' cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling of 100-475 degrees C). Well-done fried ground beef, beef steak, ham pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs and egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperatures above 225 degrees C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.

  20. A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins.

    Science.gov (United States)

    Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

    2018-06-15

    Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α 1 -acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

    Science.gov (United States)

    Kumar, C Sudheer; Swamy, Musti J

    2016-07-05

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.

  2. Dietary Intake of High-Protein Foods and Other Major Foods in Meat-Eaters, Poultry-Eaters, Fish-Eaters, Vegetarians, and Vegans in UK Biobank

    Science.gov (United States)

    2017-01-01

    Vegetarian diets are defined by the absence of meat and fish, but differences in the intake of other foods between meat-eaters and low or non-meat eaters are also important to document. We examined intakes of high-protein foods (meat, poultry, fish, legumes, nuts, vegetarian protein alternatives, dairy products, and eggs) and other major food groups (fruit, vegetables, bread, pasta, rice, snack foods, and beverages) in regular meat-eaters, low meat-eaters, poultry-eaters, fish-eaters, vegetarians, and vegans of white ethnicity participating in UK Biobank who had completed at least one web-based 24-h dietary assessment (n = 199,944). In regular meat-eaters, around 25% of total energy came from meat, fish, dairy and plant milk, cheese, yogurt, and eggs. In vegetarians, around 20% of energy came from dairy and plant milk, cheese, yoghurt, eggs, legumes, nuts, and vegetarian protein alternatives, and in vegans around 15% came from plant milk, legumes, vegetarian alternatives, and nuts. Low and non-meat eaters had higher intakes of fruit and vegetables and lower intakes of roast or fried potatoes compared to regular meat-eaters. The differences in the intakes of meat, plant-based high-protein foods, and other foods between meat-eaters and low and non-meat eaters in UK Biobank may contribute to differences in health outcomes. PMID:29207491

  3. Dietary Intake of High-Protein Foods and Other Major Foods in Meat-Eaters, Poultry-Eaters, Fish-Eaters, Vegetarians, and Vegans in UK Biobank.

    Science.gov (United States)

    Bradbury, Kathryn E; Tong, Tammy Y N; Key, Timothy J

    2017-12-02

    Vegetarian diets are defined by the absence of meat and fish, but differences in the intake of other foods between meat-eaters and low or non-meat eaters are also important to document. We examined intakes of high-protein foods (meat, poultry, fish, legumes, nuts, vegetarian protein alternatives, dairy products, and eggs) and other major food groups (fruit, vegetables, bread, pasta, rice, snack foods, and beverages) in regular meat-eaters, low meat-eaters, poultry-eaters, fish-eaters, vegetarians, and vegans of white ethnicity participating in UK Biobank who had completed at least one web-based 24-h dietary assessment ( n = 199,944). In regular meat-eaters, around 25% of total energy came from meat, fish, dairy and plant milk, cheese, yogurt, and eggs. In vegetarians, around 20% of energy came from dairy and plant milk, cheese, yoghurt, eggs, legumes, nuts, and vegetarian protein alternatives, and in vegans around 15% came from plant milk, legumes, vegetarian alternatives, and nuts. Low and non-meat eaters had higher intakes of fruit and vegetables and lower intakes of roast or fried potatoes compared to regular meat-eaters. The differences in the intakes of meat, plant-based high-protein foods, and other foods between meat-eaters and low and non-meat eaters in UK Biobank may contribute to differences in health outcomes.

  4. Structure analysis of OmpC, one of the major proteins in the outer membrane of E. coli, by high resolution electron microscopy

    International Nuclear Information System (INIS)

    Chang, C.F.

    1983-07-01

    This dissertation is concerned with the structure analysis of a pore-forming membrane protein, OmpC, which is one of the major proteins in the outer membrane of Escherichia coli. In order to obtain structural information it was necessary to develop a suitable technique for preparing two-dimensional crystalline arrays of this membrane protein in an unfixed, unstained and hydrated condition. Electron micrographs were recorded at exposures of less than 5 electrons/A 2 in order to avoid severe radiation damage. The resulting images were crystallographically averaged, in order to overcome the statistical limitations associated with the low electron exposures. The resulting images, which extend to a resolution of approx. 13.5 A, lend themselves to a natural interpretation that is consistent with the mass density of protein, water and lipid, prior data from 2-D and 3-D structure studies of negatively stained specimens at approx. = 20 A resolution, and published spectroscopic data on the peptide chain secondary structure

  5. Dietary Intake of High-Protein Foods and Other Major Foods in Meat-Eaters, Poultry-Eaters, Fish-Eaters, Vegetarians, and Vegans in UK Biobank

    Directory of Open Access Journals (Sweden)

    Kathryn E. Bradbury

    2017-12-01

    Full Text Available Vegetarian diets are defined by the absence of meat and fish, but differences in the intake of other foods between meat-eaters and low or non-meat eaters are also important to document. We examined intakes of high-protein foods (meat, poultry, fish, legumes, nuts, vegetarian protein alternatives, dairy products, and eggs and other major food groups (fruit, vegetables, bread, pasta, rice, snack foods, and beverages in regular meat-eaters, low meat-eaters, poultry-eaters, fish-eaters, vegetarians, and vegans of white ethnicity participating in UK Biobank who had completed at least one web-based 24-h dietary assessment (n = 199,944. In regular meat-eaters, around 25% of total energy came from meat, fish, dairy and plant milk, cheese, yogurt, and eggs. In vegetarians, around 20% of energy came from dairy and plant milk, cheese, yoghurt, eggs, legumes, nuts, and vegetarian protein alternatives, and in vegans around 15% came from plant milk, legumes, vegetarian alternatives, and nuts. Low and non-meat eaters had higher intakes of fruit and vegetables and lower intakes of roast or fried potatoes compared to regular meat-eaters. The differences in the intakes of meat, plant-based high-protein foods, and other foods between meat-eaters and low and non-meat eaters in UK Biobank may contribute to differences in health outcomes.

  6. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    Science.gov (United States)

    Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715

  7. Pig major acute-phase protein and haptoglobin serum concentrations correlate with PCV2 viremia and the clinical course of postweaning multisystemic wasting syndrome

    DEFF Research Database (Denmark)

    Grau-Roma, Llorenc; Heegaard, Peter M. H.; Hjulsager, Charlotte Kristiane

    2009-01-01

    -PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APP's concentrations was also assessed. Fourteen independent batches of 100 to 154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs......The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non...... and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted creteria and pigs were classified as: i)PMWS cases, ii) wasted non-PMWS cases and iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration...

  8. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

    Science.gov (United States)

    Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

  9. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    Directory of Open Access Journals (Sweden)

    Arumugam Muthu

    2016-11-01

    Full Text Available Abiotic stress in oleaginous microalgae enhances lipid accumulation and is stored in a specialised organelle called lipid droplets (LDs. Both the LDs and body are enriched with major lipid droplet protein (MLDP. It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of S. quadricauda under the salt stress of 10mM concentration is about 0.174μ and in control, the SGR is 0.241μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17. The dry biomass content also decreased drastically at 50mM salt-treated cells (129mg/L compared to control (236mg/L on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  10. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    International Nuclear Information System (INIS)

    Javee, Anand; Sulochana, Sujitha Balakrishnan; Pallissery, Steffi James; Arumugam, Muthu

    2016-01-01

    Abiotic stress in oleaginous microalgae enhances lipid accumulation, which is stored in a specialized organelle called lipid droplets (LDs). Both the LDs or lipid body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of Scenedesmus quadricauda under the salt stress of 10mM concentration is about 0.174 μ and in control, the SGR is 0.241 μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129 mg/L) compared to control (236 mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  11. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    Energy Technology Data Exchange (ETDEWEB)

    Javee, Anand [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Sulochana, Sujitha Balakrishnan [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India); Pallissery, Steffi James [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Arumugam, Muthu, E-mail: arumugam@niist.res.in [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India)

    2016-11-23

    Abiotic stress in oleaginous microalgae enhances lipid accumulation, which is stored in a specialized organelle called lipid droplets (LDs). Both the LDs or lipid body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of Scenedesmus quadricauda under the salt stress of 10mM concentration is about 0.174 μ and in control, the SGR is 0.241 μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129 mg/L) compared to control (236 mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  12. Possible Inhibitor from Traditional Chinese Medicine for the β Form of Calcium-Dependent Protein Kinase Type II in the Treatment of Major Depressive Disorder

    Directory of Open Access Journals (Sweden)

    Tzu-Chieh Hung

    2014-01-01

    Full Text Available Recently, an important topic of major depressive disorder (MDD had been published in 2013. MDD is one of the most prevalent and disabling mental disorders. Consequently, much research is being undertaken into the causes and treatment. It has been found that inhibition of the β form of calcium/calmodulin-dependent protein kinase type II (β-CaMKII can ameliorate the disorder. Upon screening the traditional Chinese medicine (TCM database by molecular docking, sengesterone, labiatic acid, and methyl 3-O-feruloylquinate were selected for molecular dynamics. After 20 ns simulation, the RMSD, total energy, and structure variation could define the protein-ligand interaction. Furthermore, sengesterone, the principle candidate compound, has been found to have an effect on the regulation of emotions and memory development. In structure variation, we find the sample functional group of important amino acids make the protein stable and have limited variation. Due to similarity of structure variations, we suggest that these compounds may have an effect on β-CaMKII and that sengesterone may have a similar efficacy as the control. However labiatic acid may be a stronger inhibitor of β-CaMKII based on the larger RMSD and variation.

  13. Correlation Between Expression of Recombinant Proteins and Abundance of H3K4Me3 on the Enhancer of Human Cytomegalovirus Major Immediate-Early Promoter.

    Science.gov (United States)

    Soo, Benjamin P C; Tay, Julian; Ng, Shirelle; Ho, Steven C L; Yang, Yuansheng; Chao, Sheng-Hao

    2017-08-01

    Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.

  14. Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

    Science.gov (United States)

    Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

    2003-10-01

    The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

  15. Effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on protein oxidation and textural properties of fish mince (Pagrosomus major) during frozen storage.

    Science.gov (United States)

    Wang, Tiantian; Li, Zhenxing; Yuan, Fangzhou; Lin, Hong; Pavase, Tushar Ramesh

    2017-03-01

    Frozen storage of minced fish is currently one of the most important techniques to maintain its functional properties. However, some deterioration does occur during frozen storage and cause quality loss. The effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on lipid and protein oxidation and textural properties of red sea bream (Pagrosomus major) during 90 days of frozen storage at -18 °C were investigated. All added antioxidants at 1 g kg -1 resulted in significantly lower thiobarbituric acid-reactive substances (TBARS) compared to the control during the 45 days of frozen storage. The antioxidants were also effective in retarding protein oxidation concerning to total sulfhydryl content and protein carbonyl content. Brown seaweed polyphenols and α-tocopherol significantly retarded the inactivation of Ca 2+ -ATPase activity during the first 45 days, whereas ascorbic acid had no such effect. The antioxidant activity showed either an invariable or decrease trend after 45 days storage. Adding antioxidants had a significant effect on the breaking force of the gels during the frozen storage period. These results indicate that brown seaweed polyphenols and α-tocopherol can be used to prevent oxidative reactions and thus maintain the structure of the gel formed by fish mince during frozen storage. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  16. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.

    Science.gov (United States)

    de Souza, Theo Luiz Ferraz; de Lima, Sheila Maria Barbosa; Braga, Vanessa L de Azevedo; Peabody, David S; Ferreira, Davis Fernandes; Bianconi, M Lucia; Gomes, Andre Marco de Oliveira; Silva, Jerson Lima; de Oliveira, Andréa Cheble

    2016-01-01

    Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro . The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

  17. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

    Directory of Open Access Journals (Sweden)

    Theo Luiz Ferraz de Souza

    2016-11-01

    Full Text Available Background Hepatitis C virus (HCV core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124 is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Methods Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. Results The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12, indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Discussion Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

  18. Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

    Science.gov (United States)

    Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

    2016-09-15

    A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection

  19. Diagnostic value of C-reactive protein to rule out infectious complications after major abdominal surgery: a systematic review and meta-analysis.

    Science.gov (United States)

    Gans, Sarah L; Atema, Jasper J; van Dieren, Susan; Groot Koerkamp, Bas; Boermeester, Marja A

    2015-07-01

    Infectious complications occur frequently after major abdominal surgery and have a major influence on patient outcome and hospital costs. A marker that can rule out postoperative infectious complications (PICs) could aid patient selection for safe and early hospital discharge. C-reactive protein (CRP) is a widely available, fast, and cheap marker that might be of value in detecting PIC. Present meta-analysis evaluates the diagnostic value of CRP to rule out PIC following major abdominal surgery, aiding patient selection for early discharge. A systematic literature search of Medline, PubMed, and Cochrane was performed identifying all prospective studies evaluating the diagnostic value of CRP after abdominal surgery. Meta-analysis was performed according to the PRISMA statement. Twenty-two studies were included for qualitative analysis of which 16 studies were eligible for meta-analysis, representing 2215 patients. Most studies analyzed the value of CRP in colorectal surgery (eight studies). The pooled negative predictive value (NPV) improved each day after surgery up to 90% at postoperative day (POD) 3 for a pooled CRP cutoff of 159 mg/L (range 92-200). Maximum predictive values for PICs were reached on POD 5 for a pooled CRP cutoff of 114 mg/L (range 48-150): a pooled sensitivity of 86% (95% confidence interval (CI) 79-91%), specificity of 86% (95% CI 75-92%), and a positive predictive value of 64% (95% CI 49-77%). The pooled sensitivity and specificity were significantly higher on POD 5 than on other PODs (p < 0.001). Infectious complications after major abdominal surgery are very unlikely in patients with a CRP below 159 mg/L on POD 3. This can aid patient selection for safe and early hospital discharge and prevent overuse of imaging.

  20. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

    Science.gov (United States)

    Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

    2004-10-05

    Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

  1. Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

    Science.gov (United States)

    Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

    2014-01-01

    Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

  2. The major egg reserve protein from the invasive apple snail Pomacea maculata is a complex carotenoprotein related to those of Pomacea canaliculata and Pomacea scalaris.

    Science.gov (United States)

    Pasquevich, M Y; Dreon, M S; Heras, H

    2014-03-01

    Snails from the genus Pomacea lay conspicuous masses of brightly colored eggs above the water. Coloration is given by carotenoproteins that also which play important roles in protection against sun radiation, stabilizing and transporting antioxidant molecules and helping to protect embryos from desiccation and predators. They seem a key acquisition, but have been little studied. Here we report the characteristics of the major carotenoprotein from Pomacea maculata and the first comparison among these egg proteins. This particle, hereafter PmPV1, represents ~52% of perivitellin fluid protein. It is a glyco-lipo-carotenoprotein responsible for the bright reddish egg coloration. With VHDL characteristics, PmPV1 apparent molecular mass is 294kDa, composed of five non-covalently bound subunits of pI 4.7-9.8 and masses between 26 and 36kDa whose N-terminal sequences were obtained. It is a glyco-lipo-carotenoprotein scarcely lipidated (strategy of Pomacea. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

    Science.gov (United States)

    Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

    2017-11-01

    MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

  4. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

    International Nuclear Information System (INIS)

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-01-01

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX

  5. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

    Science.gov (United States)

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

  6. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP with Two Different Adjuvants.

    Directory of Open Access Journals (Sweden)

    Shahneaz Ali Khan

    Full Text Available Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus. In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP antigen-based vaccine, combined with immune stimulating complex (ISC adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

  7. Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (Citrus sinensis L. Osb.).

    Science.gov (United States)

    Martins, Cristina de Paula Santos; Pedrosa, Andresa Muniz; Du, Dongliang; Gonçalves, Luana Pereira; Yu, Qibin; Gmitter, Frederick G; Costa, Marcio Gilberto Cardoso

    2015-01-01

    The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast (TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb.), the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs) were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs) based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

  8. Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (Citrus sinensis L. Osb..

    Directory of Open Access Journals (Sweden)

    Cristina de Paula Santos Martins

    Full Text Available The family of aquaporins (AQPs, or major intrinsic proteins (MIPs, includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs, tonoplast (TIPs, NOD26-like (NIPs, small basic (SIPs and unclassified X (XIPs intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb., the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

  9. Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

    Science.gov (United States)

    Wright, Douglas G; Castore, Reneau; Shi, Runhua; Mallick, Amrita; Ennis, Don G; Harrison, Lynn

    2017-03-01

    Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen

  10. Elevated maternal placental protein 13 serum levels at term of pregnancy in postpartum major hemorrhage (>1000 mLs). A prospective cohort study.

    Science.gov (United States)

    Farina, Antonio; Bernabini, Dalila; Zucchini, Cinzia; De Sanctis, Paola; Quezada, Maria Soledad; Mattioli, Mara; Rizzo, Nicola

    2017-09-01

    To compare placental protein 13 (PP13) levels in the serum of women with primary postpartum hemorrhage (PPH) with a control population. A prospective cohort study was conducted between May 2014 and May 2016 and included 435 pregnant women at term (38 weeks gestation) without any known risk factor and with normal labor. Multiples of median (MoM) were used to evaluate differences of the PP13 values between cases and controls. PP13 concentrations were adjusted for maternal and neonatal weight. Multivariable analysis was used to detect independent contribution of predictors of PPH. Fifteen had a major PPH >1000 mLs and represented the cases of the study. They were matched with 399 controls. Twenty-one patients who had a minor PPH (500-1000 mLs) were excluded. The mean observed rank in the PPH group was higher than that of controls (28.5 vs 13.5, P-value=.01). PP13 MoM values adjusted for maternal weight were higher than expected being 1.44±0.45 in PPH cases and 1.00±0.59 in controls (P-value .008). This difference was still significant even after adjustment for neonatal weight that represented a confounding variable. Higher PP13 levels are independently associated with major PPH >1000 mLs. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. BALB/c Mice Vaccinated with Leishmania major Ribosomal Proteins Extracts Combined with CpG Oligodeoxynucleotides Become Resistant to Disease Caused by a Secondary Parasite Challenge

    Directory of Open Access Journals (Sweden)

    Laura Ramírez

    2010-01-01

    Full Text Available Leishmaniasis is an increasing public health problem and effective vaccines are not currently available. We have previously demonstrated that vaccination with ribosomal proteins extracts administered in combination of CpG oligodeoxynucleotides protects susceptible BALB/c mice against primary Leishmania major infection. Here, we evaluate the long-term immunity to secondary infection conferred by this vaccine. We show that vaccinated and infected BALB/c mice were able to control a secondary Leishmania major challenge, since no inflammation and very low number of parasites were observed in the site of reinfection. In addition, although an increment in the parasite burden was observed in the draining lymph nodes of the primary site of infection we did not detected inflammatory lesions at that site. Resistance against reinfection correlated to a predominant Th1 response against parasite antigens. Thus, cell cultures established from spleens and the draining lymph node of the secondary site of infection produced high levels of parasite specific IFN-γ in the absence of IL-4 and IL-10 cytokine production. In addition, reinfected mice showed a high IgG2a/IgG1 ratio for anti-Leishmania antibodies. Our results suggest that ribosomal vaccine, which prevents pathology in a primary challenge, in combination with parasite persistence might be effective for long-term maintenance of immunity.

  12. High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    Directory of Open Access Journals (Sweden)

    Weiguo Hou

    2018-05-01

    Full Text Available Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length encoding the cyanophage gp23 major capsid protein (MCP. Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92% belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

  13. Effects of feeding canola meal or wheat dried distillers grains with solubles as a major protein source in low- or high-crude protein diets on ruminal fermentation, omasal flow, and production in cows.

    Science.gov (United States)

    Mutsvangwa, T; Kiran, D; Abeysekara, S

    2016-02-01

    The objective of this study was to determine the effects of feeding canola meal (CM) or wheat dried distillers grains with solubles (W-DDGS) as the major source of protein in diets varying in crude protein (CP) content on ruminal fermentation, microbial protein production, omasal nutrient flow, and production performance in lactating dairy cows. Eight lactating dairy cows were used in a replicated 4×4 Latin square design with 29-d periods (21 d of dietary adaptation and 8 d of measurements) and a 2×2 factorial arrangement of dietary treatments. Four cows in 1 Latin square were ruminally cannulated to allow ruminal and omasal sampling. The treatment factors were (1) source of supplemental protein (CM vs. W-DDGS) and (2) dietary CP content (15 vs. 17%; DM basis). Diets contained 50% forage and 50% concentrate, and were fed twice daily at 0900 and 1600 h as total mixed rations for ad libitum intake. Dry matter intake and milk yield were unaffected by dietary treatments; however, milk yield in cows that were fed CM was numerically greater (+1.1 kg/d) when compared with cows fed W-DDGS. Feeding CM increased milk lactose content compared with feeding W-DDGS. Milk urea nitrogen and ruminal NH3-N concentrations were greater in cows fed the high-CP compared with those fed the low-CP diet. The rumen-degradable protein supply was greater in cows fed the high-CP when compared with those fed the low-CP diet when diets contained CM, whereas rumen-degradable protein supply was lower in cows fed the high-CP when compared with those fed the low-CP diet when diets contained W-DDGS. Total N flow at the omasal canal was not affected by diet; however, omasal flow of NH3-N was greater in cows fed CM when compared with those fed W-DDGS. The rumen-undegradable protein supply was greater in cows fed the low-CP when compared with those fed the high-CP diet when diets contained CM, whereas rumen-undegradable protein supply was lower in cows fed the low-CP when compared with those fed the

  14. Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins

    International Nuclear Information System (INIS)

    Popham, D.L.; Sengupta, S.; Setlow, P.

    1995-01-01

    Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores. Spores lacking the two major α/β-type small, acid-soluble proteins (SASP) (termed a α - β - spores) have the same core water content as do wild-type spores, but α - β - dacB spores had more core water than did dacB spores. The resistance of α - β - , α - β - dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide. These data (1) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of α/β-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (2) suggest that binding of αβ-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (3) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (4) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by α/β-type SASP. 19 refs., 2 figs., 5 tabs

  15. A Major Facilitator Superfamily protein encoded by TcMucK gene is not required for cuticle pigmentation, growth and development in Tribolium castaneum.

    Science.gov (United States)

    Mun, Seulgi; Noh, Mi Young; Osanai-Futahashi, Mizuko; Muthukrishnan, Subbaratnam; Kramer, Karl J; Arakane, Yasuyuki

    2014-06-01

    Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T. castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene during development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down

  16. Major depression

    Science.gov (United States)

    Depression - major; Depression - clinical; Clinical depression; Unipolar depression; Major depressive disorder ... providers do not know the exact causes of depression. It is believed that chemical changes in the ...

  17. Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein.

    Science.gov (United States)

    Looby, Mairead; Ibarra, Neysi; Pierce, James J; Buckley, Kevin; O'Donovan, Eimear; Heenan, Mary; Moran, Enda; Farid, Suzanne S; Baganz, Frank

    2011-01-01

    This study describes the application of quality by design (QbD) principles to the development and implementation of a major manufacturing process improvement for a commercially distributed therapeutic protein produced in Chinese hamster ovary cell culture. The intent of this article is to focus on QbD concepts, and provide guidance and understanding on how the various components combine together to deliver a robust process in keeping with the principles of QbD. A fed-batch production culture and a virus inactivation step are described as representative examples of upstream and downstream unit operations that were characterized. A systematic approach incorporating QbD principles was applied to both unit operations, involving risk assessment of potential process failure points, small-scale model qualification, design and execution of experiments, definition of operating parameter ranges and process validation acceptance criteria followed by manufacturing-scale implementation and process validation. Statistical experimental designs were applied to the execution of process characterization studies evaluating the impact of operating parameters on product quality attributes and process performance parameters. Data from process characterization experiments were used to define the proven acceptable range and classification of operating parameters for each unit operation. Analysis of variance and Monte Carlo simulation methods were used to assess the appropriateness of process design spaces. Successful implementation and validation of the process in the manufacturing facility and the subsequent manufacture of hundreds of batches of this therapeutic protein verifies the approaches taken as a suitable model for the development, scale-up and operation of any biopharmaceutical manufacturing process. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  18. Major Vault Protein, a Candidate Gene in 16p11.2 Microdeletion Syndrome, Is Required for the Homeostatic Regulation of Visual Cortical Plasticity.

    Science.gov (United States)

    Ip, Jacque P K; Nagakura, Ikue; Petravicz, Jeremy; Li, Keji; Wiemer, Erik A C; Sur, Mriganka

    2018-04-18

    Microdeletion of a region in chromosome 16p11.2 increases susceptibility to autism. Although this region contains exons of 29 genes, disrupting only a small segment of the region, which spans five genes, is sufficient to cause autistic traits. One candidate gene in this critical segment is MVP , which encodes for the major vault protein (MVP) that has been implicated in regulation of cellular transport mechanisms. MVP expression levels in MVP +/- mice closely phenocopy those of 16p11.2 mutant mice, suggesting that MVP +/- mice may serve as a model of MVP function in 16p11.2 microdeletion. Here we show that MVP regulates the homeostatic component of ocular dominance (OD) plasticity in primary visual cortex. MVP +/- mice of both sexes show impairment in strengthening of open-eye responses after several days of monocular deprivation (MD), whereas closed-eye responses are weakened as normal, resulting in reduced overall OD plasticity. The frequency of miniature EPSCs (mEPSCs) in pyramidal neurons is decreased in MVP +/- mice after extended MD, suggesting a reduction of functional synapses. Correspondingly, upregulation of surface GluA1 AMPA receptors is reduced in MVP +/- mice after extended MD, and is accompanied by altered expression of STAT1 and phosphorylated ERK, which have been previously implicated in OD plasticity. Normalization of STAT1 levels by introducing STAT1 shRNA rescues surface GluA1 and open-eye responses, implicating STAT1 as a downstream effector of MVP. These findings demonstrate a specific role for MVP as a key molecule influencing the homeostatic component of activity-dependent synaptic plasticity, and potentially the corresponding phenotypes of 16p11.2 microdeletion syndrome. SIGNIFICANCE STATEMENT Major vault protein (MVP), a candidate gene in 16p11.2 microdeletion syndrome, has been implicated in the regulation of several cellular processes including transport mechanisms and scaffold signaling. However, its role in brain function and

  19. Urinary Vitamin D Binding Protein and KIM-1 Are Potent New Biomarkers of Major Adverse Renal Events in Patients Undergoing Coronary Angiography.

    Directory of Open Access Journals (Sweden)

    Lyubov Chaykovska

    Full Text Available Vitamin-D-binding protein (VDBP is a low molecular weight protein that is filtered through the glomerulus as a 25-(OH vitamin D 3/VDBP complex. In the normal kidney VDBP is reabsorbed and catabolized by proximal tubule epithelial cells reducing the urinary excretion to trace amounts. Acute tubular injury is expected to result in urinary VDBP loss. The purpose of our study was to explore the potential role of urinary VDBP as a biomarker of an acute renal damage.We included 314 patients with diabetes mellitus or mild renal impairment undergoing coronary angiography and collected blood and urine before and 24 hours after the CM application. Patients were followed for 90 days for the composite endpoint major adverse renal events (MARE: need for dialysis, doubling of serum creatinine after 90 days, unplanned emergency rehospitalization or death.Increased urine VDBP concentration 24 hours after contrast media exposure was predictive for dialysis need (no dialysis: 113.06 ± 299.61 ng/ml, n = 303; need for dialysis: 613.07 ± 700.45 ng/ml, n = 11, Mean ± SD, p<0.001, death (no death during follow-up: 121.41 ± 324.45 ng/ml, n = 306; death during follow-up: 522.01 ± 521.86 ng/ml, n = 8; Mean ± SD, p<0.003 and MARE (no MARE: 112.08 ± 302.00 ng/ml, n = 298; MARE: 506.16 ± 624.61 ng/ml, n = 16, Mean ± SD, p<0.001 during the follow-up of 90 days after contrast media exposure. Correction of urine VDBP concentrations for creatinine excretion confirmed its predictive value and was consistent with increased levels of urinary Kidney Injury Molecule-1 (KIM-1 and baseline plasma creatinine in patients with above mentioned complications. The impact of urinary VDBP and KIM-1 on MARE was independent of known CIN risk factors such as anemia, preexisting renal failure, preexisting heart failure, and diabetes.Urinary VDBP is a promising novel biomarker of major contrast induced nephropathy-associated events 90 days after contrast media exposure.

  20. Vast diversity of prokaryotic virus genomes encoding double jelly-roll major capsid proteins uncovered by genomic and metagenomic sequence analysis.

    Science.gov (United States)

    Yutin, Natalya; Bäckström, Disa; Ettema, Thijs J G; Krupovic, Mart; Koonin, Eugene V

    2018-04-10

    Analysis of metagenomic sequences has become the principal approach for the study of the diversity of viruses. Many recent, extensive metagenomic studies on several classes of viruses have dramatically expanded the visible part of the virosphere, showing that previously undetected viruses, or those that have been considered rare, actually are important components of the global virome. We investigated the provenance of viruses related to tail-less bacteriophages of the family Tectiviridae by searching genomic and metagenomics sequence databases for distant homologs of the tectivirus-like Double Jelly-Roll major capsid proteins (DJR MCP). These searches resulted in the identification of numerous genomes of virus-like elements that are similar in size to tectiviruses (10-15 kilobases) and have diverse gene compositions. By comparison of the gene repertoires, the DJR MCP-encoding genomes were classified into 6 distinct groups that can be predicted to differ in reproduction strategies and host ranges. Only the DJR MCP gene that is present by design is shared by all these genomes, and most also encode a predicted DNA-packaging ATPase; the rest of the genes are present only in subgroups of this unexpectedly diverse collection of DJR MCP-encoding genomes. Only a minority encode a DNA polymerase which is a hallmark of the family Tectiviridae and the putative family "Autolykiviridae". Notably, one of the identified putative DJR MCP viruses encodes a homolog of Cas1 endonuclease, the integrase involved in CRISPR-Cas adaptation and integration of transposon-like elements called casposons. This is the first detected occurrence of Cas1 in a virus. Many of the identified elements are individual contigs flanked by inverted or direct repeats and appear to represent complete, extrachromosomal viral genomes, whereas others are flanked by bacterial genes and thus can be considered as proviruses. These contigs come from metagenomes of widely different environments, some dominated by

  1. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  2. Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

    Directory of Open Access Journals (Sweden)

    Kriangsuk Srijaruskul

    Full Text Available Stenotrophomonas maltophilia MfsA (Smlt1083 is an efflux pump in the major facilitator superfamily (MFS. Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084 is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.

  3. Association between abnormal serum myelin-specific protein levels and white matter integrity in first-episode and drug-naïve patients with major depressive disorder.

    Science.gov (United States)

    Jiang, Linling; Cheng, Yuqi; Jiang, Hongyan; Xu, Jian; Lu, Jin; Shen, Zonglin; Lu, Yi; Liu, Fang; Li, Luqiong; Xu, Xiufeng

    2018-05-01

    Although the structural abnormalities of white matter (WM) have been described in patients with major depressive disorder (MDD), the neuropathological changes remain unclear. The current study aimed to investigate the myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG) levels and their correlations with WM integrity in first-episode, drug-naïve MDD patients. We obtained diffusion tensor images of 102 first-episode, drug-naïve MDD patients and 81 age- and sex-matched controls. Serum MOG and MAG levels of all participants were measured and compared between the two groups. The correlations between WM integrity and MOG and MAG levels were examined. MOG and MAG serum levels were significantly higher in MDD patients than in controls. Patients with MDD also showed decreased fractional anisotropy (FA) and axial diffusivity in the WM of the bilateral thalamus, right hippocampus, right temporal lobe, and left pulvinar. At the whole-brain level, no regions showed any correlations of diffusivity parameters with MOG or MAG levels in healthy subjects. However, we observed two-way correlations between the MOG and MAG levels and the FA and mean diffusivity values in the WM of the left middle frontal lobe, right inferior parietal lobe, and right supplementary motor area in MDD patients. Further investigation with a larger sample size and longitudinal studies are required to better understand the neuropathology of WM integrity in MDD. Our findings represent the first evidence of a relationship between abnormal serum myelin-specific protein levels and impaired WM integrity, which may help to better understand the neurobiological mechanisms of MDD. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Effect of agomelatine treatment on C-reactive protein levels in patients with major depressive disorder: an exploratory study in "real-world," everyday clinical practice.

    Science.gov (United States)

    De Berardis, Domenico; Fornaro, Michele; Orsolini, Laura; Iasevoli, Felice; Tomasetti, Carmine; de Bartolomeis, Andrea; Serroni, Nicola; De Lauretis, Ida; Girinelli, Gabriella; Mazza, Monica; Valchera, Alessandro; Carano, Alessandro; Vellante, Federica; Matarazzo, Ilaria; Perna, Giampaolo; Martinotti, Giovanni; Di Giannantonio, Massimo

    2017-08-01

    Agomelatine is a newer antidepressant but, to date, no studies have been carried out investigating its effects on C-reactive protein (CRP) levels in major depressive disorder (MDD) before and after treatment. The present study aimed (i) to investigate the effects of agomelatine treatment on CRP levels in a sample of patients with MDD and (ii) to investigate if CRP variations were correlated with clinical improvement in such patients. 30 adult outpatients (12 males, 18 females) with a Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) diagnosis of MDD were recruited in "real-world," everyday clinical practice and treated with a flexible dose of agomelatine for 12 weeks. The Hamilton Rating Scale for Depression (HAM-D) and the Snaith-Hamilton Pleasure Scale (SHAPS) were used to evaluate depressive symptoms and anhedonia, respectively. Moreover, serum CRP was measured at baseline and after 12 weeks of treatment. Agomelatine was effective in the treatment of MDD, with a significant reduction in HAM-D and SHAPS scores from baseline to endpoint. CRP levels were reduced in the whole sample, with remitters showing a significant difference in CRP levels after 12 weeks of agomelatine. A multivariate stepwise linear regression analysis showed that higher CRP level variation was associated with higher baseline HAM-D scores, controlling for age, gender, smoking, BMI, and agomelatine dose. Agomelatine's antidepressant properties were associated with a reduction in circulating CRP levels in MDD patients who achieved remission after 12 weeks of treatment. Moreover, more prominent CRP level variation was associated with more severe depressive symptoms at baseline.

  5. Comparison of clinical performance of antigen basedenzyme immunoassay (EIA and major outer membrane protein (MOMP-PCR for detection of genital Chlamydia trachomatis infection

    Directory of Open Access Journals (Sweden)

    Mahmoud Nateghi Rostami

    2016-06-01

    Full Text Available Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA and major outer membrane protein (MOMP-polymerase chain reaction (PCR for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV and negative predictive values (NPV were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14% cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMPPCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48. Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  6. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

    Science.gov (United States)

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-06-01

    Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  7. Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine.

    Science.gov (United States)

    Khan, Shahneaz Ali; Polkinghorne, Adam; Waugh, Courtney; Hanger, Jon; Loader, Jo; Beagley, Kenneth; Timms, Peter

    2016-02-03

    The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

    Science.gov (United States)

    Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

    2003-01-01

    Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

  9. A quantitative model of the major pathways for radiation-induced DNA double-strand break repair

    International Nuclear Information System (INIS)

    Belov, O.V.; Krasavin, E.A.; Lyashko, M.S.; Batmunkh, M.; Sweilam, N.H.

    2014-01-01

    We have developed a model approach to simulate the major pathways of DNA double-strand break (DSB) repair in mammalian and human cells. The proposed model shows a possible mechanistic explanation of the basic regularities of DSB processing through the nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). It reconstructs the time-courses of radiation-induced foci specific to particular repair processes including the major intermediate stages. The model is validated for ionizing radiations of a wide range of linear energy transfer (0.2-236 keV/μm) including a relatively broad spectrum of heavy ions. The appropriate set of reaction rate constants was suggested to satisfy the kinetics of DSB rejoining for the considered types of exposure. The simultaneous assessment of three repair pathways allows one to describe their possible biological relations in response to radiation. With the help of the proposed approach, we reproduce several experimental data sets on γ-H2AX foci remaining in different types of cells including those defective in NHEJ, HR, or SSA functions.

  10. Activated protein C plays no major roles in the inhibition of coagulation or increased fibrinolysis in acute coagulopathy of trauma-shock: a systematic review.

    Science.gov (United States)

    Gando, Satoshi; Mayumi, Toshihiko; Ukai, Tomohiko

    2018-01-01

    The pathophysiological mechanisms of acute coagulopathy of trauma-shock (ACOTS) are reported to include activated protein C-mediated suppression of thrombin generation via the proteolytic inactivation of activated Factor V (FVa) and FVIIIa; an increased fibrinolysis via neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C. The aims of this study are to review the evidences for the role of activated protein C in thrombin generation and fibrinolysis and to validate the diagnosis of ACOTS based on the activated protein C dynamics. We conducted systematic literature search (2007-2017) using PubMed, the Cochrane Database of Systematic Reviews (CDSR), and the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical studies on trauma that measured activated protein C or the circulating levels of activated protein C-related coagulation and fibrinolysis markers were included in our study. Out of 7613 studies, 17 clinical studies met the inclusion criteria. The levels of activated protein C in ACOTS were inconsistently decreased, showed no change, or were increased in comparison to the control groups. Irrespective of the activated protein C levels, thrombin generation was always preserved or highly elevated. There was no report on the activated protein C-mediated neutralization of PAI-1 with increased fibrinolysis. No included studies used unified diagnostic criteria to diagnose ACOTS and those studies also used different terms to refer to the condition known as ACOTS. None of the studies showed direct cause and effect relationships between activated protein C and the suppression of coagulation and increased fibrinolysis. No definitive diagnostic criteria or unified terminology have been established for ACOTS based on the activated protein C dynamics.

  11. Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates

    DEFF Research Database (Denmark)

    Krintel, Christian; Osmark, Peter; Larsen, Martin Røssel

    2008-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well...

  12. Listeria-vectored vaccine expressing the Mycobacterium tuberculosis 30 kDa major secretory protein via the constitutively active prfA* regulon boosts BCG efficacy against tuberculosis.

    Science.gov (United States)

    Jia, Qingmei; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

    2017-06-19

    A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis (Mtb) 30 kDa major secretory protein (r30/Ag85B) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated Lm vectors, rLm Δ actA (LmI), rLm Δ actA Δ inlB (LmII), and rLm Δ actA Δ inlB prfA * (LmIII), we constructed five rLm30 vaccine candidates expressing the r30 linked in-frame to the Lm Listeriolycin O signal sequence and driven by the hly promoter (h30) or linked in-frame to the ActA N-terminus and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm expressing r30 via a constitutively active prfA * regulon (rLmIII/a30) expressed the greatest amount of r30 in broth culture, all five rLm vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T-cells expressing the three cytokines of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) ( P vaccines were generally more potent booster vaccines than r30 in adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized Mtb ( P <0.01). Copyright © 2017 American Society for Microbiology.

  13. Genome-Wide Characterization of Major Intrinsic Proteins in Four Grass Plants and Their Non-Aqua Transport Selectivity Profiles with Comparative Perspective.

    Directory of Open Access Journals (Sweden)

    Abul Kalam Azad

    Full Text Available Major intrinsic proteins (MIPs, commonly known as aquaporins, transport not only water in plants but also other substrates of physiological significance and heavy metals. In most of the higher plants, MIPs are divided into five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs. Herein, we identified 68, 42, 38 and 28 full-length MIPs, respectively in the genomes of four monocot grass plants, specifically Panicum virgatum, Setaria italica, Sorghum bicolor and Brachypodium distachyon. Phylogenetic analysis showed that the grass plants had only four MIP subfamilies including PIPs, TIPs, NIPs and SIPs without XIPs. Based on structural analysis of the homology models and comparing the primary selectivity-related motifs [two NPA regions, aromatic/arginine (ar/R selectivity filter and Froger's positions (FPs] of all plant MIPs that have been experimentally proven to transport non-aqua substrates, we predicted the transport profiles of all MIPs in the four grass plants and also in eight other plants. Groups of MIP subfamilies based on ar/R selectivity filter and FPs were linked to the non-aqua transport profiles. We further deciphered the substrate selectivity profiles of the MIPs in the four grass plants and compared them with their counterparts in rice, maize, soybean, poplar, cotton, Arabidopsis thaliana, Physcomitrella patens and Selaginella moellendorffii. In addition to two NPA regions, ar/R filter and FPs, certain residues, especially in loops B and C, contribute to the functional distinctiveness of MIP groups. Expression analysis of transcripts in different organs indicated that non-aqua transport was related to expression of MIPs since most of the unexpressed MIPs were not predicted to facilitate the transport of non-aqua molecules. Among all MIPs in every plant, TIP (BdTIP1;1, SiTIP1;2, SbTIP2;1 and PvTIP1;2 had the overall highest mean expression. Our study generates significant information for understanding the diversity, evolution, non

  14. Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

    Science.gov (United States)

    Lawson, D M; Williams, C E; Mitchenall, L A; Pau, R N

    1998-12-15

    . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

  15. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs...

  16. Major Links.

    Science.gov (United States)

    Henderson, Tona

    1995-01-01

    Provides electronic mail addresses for resources and discussion groups related to the following academic majors: art, biology, business, chemistry, computer science, economics, health sciences, history, literature, math, music, philosophy, political science, psychology, sociology, and theater. (AEF)

  17. Major Roads

    Data.gov (United States)

    Minnesota Department of Natural Resources — This data set contains roadway centerlines for major roads (interstates and trunk highways) found on the USGS 1:24,000 mapping series. These roadways are current...

  18. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

    OpenAIRE

    Theo Luiz Ferraz de Souza; Sheila Maria Barbosa de Lima; Vanessa L. de Azevedo Braga; David S. Peabody; Davis Fernandes Ferreira; M. Lucia Bianconi; Andre Marco de Oliveira Gomes; Jerson Lima Silva; Andréa Cheble de Oliveira

    2016-01-01

    Background Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specific...

  19. β-Galactosidase treatment is a common first-stage modification of the three major subtypes of Gc protein to GcMAF.

    Science.gov (United States)

    Uto, Yoshihiro; Yamamoto, Syota; Mukai, Hirotaka; Ishiyama, Noriko; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Hori, Hitoshi

    2012-06-01

    The 1f1f subtype of the group-specific component (Gc) protein is converted into Gc protein-derived macrophage-activating factor (GcMAF) by enzymatic processing with β-galactosidase and sialidase. We previously demonstrated that preGc(1f1f)MAF, a full Gc(1f1f) protein otherwise lacking a galactosyl moiety, can be converted to GcMAF by treatment with mouse peritoneal fluid. Here, we investigated the effects of the β-galactosidase-treated 1s1s and 22 subtypes of Gc protein (preGc(1s1s)MAF and preGc₂₂MAF) on the phagocytic activation of mouse peritoneal macrophages. We demonstrated the presence of Gal-GalNAc disaccharide sugar structures in the Gc(1s1s) protein by western blotting using peanut agglutinin and Helix pomatia agglutinin lectin. We also found that preGc(1s1s)MAF and preGc₂₂MAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages in the presence and absence of mouse peritoneal fluid. We demonstrate that preGc(1s1s)MAF and preGc₂₂MAF proteins can be used as effective macrophage activators.

  20. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  1. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    International Nuclear Information System (INIS)

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-01-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus

  2. Formation of high-molecular-weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil major basic protein

    DEFF Research Database (Denmark)

    Kløverpris, Søren; Skov, Louise Lind; Glerup, Simon

    2013-01-01

    compared to monomeric AGT and the proMBP-AGT complex. Furthermore, we have used recombinant proteins to analyse the formation of the proMBP-PAPP-A and the proMBP-AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially...... on the redox potential, potentially important for the relative amounts of the complexes in vivo. These findings may be important physiologically, since the biochemical properties of the proteins change as a consequence of complex formation....

  3. Comparison of Maize Silage-based Diets for Dairy Cows Containing Extruded Rapeseed Cake or Extruded Full-fat Soybean as Major Protein Sources

    Directory of Open Access Journals (Sweden)

    Jiří Třináctý

    2016-01-01

    Full Text Available The trial was carried out on four Holstein cows with initial milk yield of 27.3 ± 1.7 kg.day−1. Cows were divided into two groups – the first was fed a diet based on extruded rapeseed cake (D-ERC, the second one was fed a diet based on extruded full-fat soybean (D-EFFS, both diets contained maize silage and meadow hay. The experiment was divided into 4 periods of 42 days. Intake of dry matter, crude protein and NEL was not affected by the treatment (P > 0.05 while the intake of PDIA, PDIN and PDIE was lower in D-ERC than in D-EFFS (P < 0.05. Milk yield in D-ERC (22.6 kg.d−1 was lower than in D-EFFS (24.7 kg.d−1, P < 0.001 while concentration of milk fat and protein were reverse (P < 0.05. Smaller portion of essential AADI in crude protein intake (CPI in D-ERC resulted in lower efficiency of CPI utilization for milk protein synthesis in comparison to D-EFFS being 313 and 327 g.kg−1, respectively (P < 0.01. Concentration of AA in blood plasma was not affected by the type of diet except of His and Ile that were higher in D-EFFS (P < 0.01.

  4. Ribosomal dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in Lactococcus lactis

    DEFF Research Database (Denmark)

    Breuner, Anne; Frees, Dorte; Varmanen, Pekka

    2016-01-01

    R, CcpA, ArgR and AhrC regulons, while protein synthesis stopped due to an extremely low GTP concentration emerging a few minutes after glucose depletion. The yfiA deletion mutant exhibited a longer lag phase upon replenishment of glucose and a faster death rate after prolonged starvation supporting...

  5. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    Directory of Open Access Journals (Sweden)

    M Emmy M Dolman

    Full Text Available Tumor cells might resist therapy with ionizing radiation (IR by non-homologous end-joining (NHEJ of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK. The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  6. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    Science.gov (United States)

    Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  7. Effects of protein hydrolysates supplementation in low fish meal diets on growth performance, innate immunity and disease resistance of red sea bream Pagrus major.

    Science.gov (United States)

    Khosravi, Sanaz; Rahimnejad, Samad; Herault, Mikaël; Fournier, Vincent; Lee, Cho-Rong; Dio Bui, Hien Thi; Jeong, Jun-Bum; Lee, Kyeong-Jun

    2015-08-01

    This study was conducted to evaluate the supplemental effects of three different types of protein hydrolysates in a low fish meal (FM) diet on growth performance, feed utilization, intestinal morphology, innate immunity and disease resistance of juvenile red sea bream. A FM-based diet was used as a high fish meal diet (HFM) and a low fish meal (LFM) diet was prepared by replacing 50% of FM by soy protein concentrate. Three other diets were prepared by supplementing shrimp, tilapia or krill hydrolysate to the LFM diet (designated as SH, TH and KH, respectively). Triplicate groups of fish (4.9 ± 0.1 g) were fed one of the test diets to apparent satiation twice daily for 13 weeks and then challenged by Edwardsiella tarda. At the end of the feeding trial, significantly (P red sea bream. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1989-01-01

    We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage

  9. Identification and quantification of major maillard cross-links in human serum albumin and lens protein. Evidence for glucosepane as the dominant compound.

    Science.gov (United States)

    Biemel, Klaus M; Friedl, D Alexander; Lederer, Markus O

    2002-07-12

    Glycation reactions leading to protein modifications (advanced glycation end products) contribute to various pathologies associated with the general aging process and long term complications of diabetes. However, only few relevant compounds have so far been detected in vivo. We now report on the first unequivocal identification of the lysine-arginine cross-links glucosepane 5, DOGDIC 6, MODIC 7, and GODIC 8 in human material. For their accurate quantification by coupled liquid chromatography-electrospray ionization mass spectrometry, (13)C-labeled reference compounds were synthesized independently. Compounds 5-8 are formed via the alpha-dicarbonyl compounds N(6)-(2,3-dihydroxy-5,6-dioxohexyl)-l-lysinate (1a,b), 3-deoxyglucosone (), methylglyoxal (), and glyoxal (), respectively. The protein-bound dideoxyosone 1a,b seems to be of prime significance for cross-linking because it presumably is not detoxified by mammalian enzymes as readily as 2-4. Hence, the follow-up product glucosepane 5 was found to be the dominant compound. Up to 42.3 pmol of 5/mg of protein was identified in human serum albumin of diabetics; the level of 5 correlates markedly with the glycated hemoglobin HbA(1c). In the water-insoluble fraction of lens proteins from normoglycemics, concentration of 5 ranges between 132.3 and 241.7 pmol/mg. The advanced glycoxidation end product GODIC 8 is elevated significantly in brunescent lenses, indicating enhanced oxidative stress in this material. Compounds 5-8 thus appear predestined as markers for pathophysiological processes.

  10. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

    OpenAIRE

    Kristie, T M; Roizman, B

    1986-01-01

    Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

  11. The TolC protein of Legionella pneumophila plays a major role in multi-drug resistance and the early steps of host invasion.

    Directory of Open Access Journals (Sweden)

    Mourad Ferhat

    Full Text Available Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

  12. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

  13. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

    NARCIS (Netherlands)

    Mueller, Nancy; Berkhout, Ben; Das, Atze T.

    2015-01-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

  14. Detection of Catalase as a major protein target of the lipid peroxidation product 4-HNE and the lack of its genetic association as a risk factor in SLE

    Directory of Open Access Journals (Sweden)

    Matsumoto Hiroyuki

    2008-07-01

    Full Text Available Abstract Background Systemic lupus erythematosus (SLE is a multifactorial disorder characterized by the presence of autoantibodies. We and others have implicated free radical mediated peroxidative damage in the pathogenesis of SLE. Since harmful free radical products are formed during this oxidative process, including 4-hydroxy 2-nonenol (4-HNE and malondialdehyde (MDA, we hypothesized that specific HNE-protein adducts would be present in SLE red blood cell (RBC membranes. Catalase is located on chromosome 11p13 where linkage analysis has revealed a marker in the same region of the genome among families with thrombocytopenia, a clinical manifestation associated with severe lupus in SLE affected pedigrees. Moreover, SLE afflicts African-Americans three times more frequently than their European-American counterparts. Hence we investigated the effects of a genetic polymorphism of catalase on risk and severity of SLE in 48 pedigrees with African American ancestry. Methods Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS analysis was used to identify the protein modified by HNE, following Coomassie staining to visualize the bands on the acrylamide gels. Genotyping analysis for the C → T, -262 bp polymorphism in the promoter region of catalase was performed by PCR-RFLP and direct PCR-sequencing. We used a "pedigree disequilibrium test" for the family based association analysis, implemented in the PDT program to analyze the genotyping results. Results We found two proteins to be HNE-modified, migrating around 80 and 50 kD respectively. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS analysis of the Coomassie stained 80 kD band revealed that the target of HNE modification was catalase, a protein shown to associate with RBC membrane proteins. All the test statistics carried out on the genotyping analysis for the

  15. Silencing the Odorant Binding Protein RferOBP1768 Reduces the Strong Preference of Palm Weevil for the Major Aggregation Pheromone Compound Ferrugineol

    Directory of Open Access Journals (Sweden)

    Binu Antony

    2018-03-01

    Full Text Available In insects, perception of the environment—food, mates, and prey—is mainly guided by chemical signals. The dynamic process of signal perception involves transport to odorant receptors (ORs by soluble secretory proteins, odorant binding proteins (OBPs, which form the first stage in the process of olfactory recognition and are analogous to lipocalin family proteins in vertebrates. Although OBPs involved in the transport of pheromones to ORs have been functionally identified in insects, there is to date no report for Coleoptera. Furthermore, there is a lack of information on olfactory perception and the molecular mechanism by which OBPs participate in the transport of aggregation pheromones. We focus on the red palm weevil (RPW Rhynchophorus ferrugineus, the most devastating quarantine pest of palm trees worldwide. In this work, we constructed libraries of all OBPs and selected antenna-specific and highly expressed OBPs for silencing through RNA interference. Aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol and 4-methyl-5-nonanone (ferruginone, and a kairomone, ethyl acetate, were then sequentially presented to individual RPWs. The results showed that antenna-specific RferOBP1768 aids in the capture and transport of ferrugineol to ORs. Silencing of RferOBP1768, which is responsible for pheromone binding, significantly disrupted pheromone communication. Study of odorant perception in palm weevil is important because the availability of literature regarding the nature and role of olfactory signaling in this insect may reveal likely candidates representative of animal olfaction and, more generally, of molecular recognition. Knowledge of OBPs recognizing the specific pheromone ferrugineol will allow for designing biosensors for the detection of this key compound in weevil monitoring in date palm fields.

  16. KIN17, XPC, DNA-PKCS and XRCC4 proteins in the cellular response to DNA damages. Relations between nucleotide excision repair and non-homologous end joining in a human syn-genic model

    International Nuclear Information System (INIS)

    Despras, Emmanuelle

    2006-01-01

    The response to genotoxic stress involves many cellular factors in a complex network of mechanisms that aim to preserve the genetic integrity of the organism. These mechanisms enclose the detection and repair of DNA lesions, the regulation of transcription and replication and, eventually, the setting of cell death. Among the nuclear proteins involved in this response, kin17 proteins are zinc-finger proteins conserved through evolution and activated by ultraviolet (UV) or ionizing radiations (IR). We showed that human kin17 protein (HSAkin17) is found in the cell under a soluble form and a form tightly anchored to nuclear structures. A fraction of HSAkin17 protein is directly associated with chromatin. HSAkin17 protein is recruited to nuclear structures 24 hours after treatment with various agents inducing DNA double-strand breaks (DSB) and/or replication forks blockage. Moreover, the reduction of total HSAkin17 protein level sensitizes RKO cells to IR. We also present evidence for the involvement of HSAkin17 protein in DNA replication. This hypothesis was further confirmed by the biochemical demonstration of its belonging to the replication complex. HSAkin17 protein could link DNA replication and DNA repair, a defect in the HSAkin17 pathway leading to an increased radiosensitivity. In a second part, we studied the interactions between two DNA repair mechanisms: nucleotide excision repair (NER) and non-homologous end joining (NHEJ). NER repairs a wide variety of lesions inducing a distortion of the DNA double helix including UV-induced pyrimidine dimers. NHEJ allows the repair of DSB by direct joining of DNA ends. We used a syn-genic model for DNA repair defects based on RNA interference developed in the laboratory. Epstein-Barr virus-derived vectors (pEBV) allow long-term expression of siRNA and specific extinction of the targeted gene. The reduction of the expression of genes involved in NER (XPA and XPC) or NHEJ (DNA-PKcs and XRCC4) leads to the expected

  17. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins.

    Science.gov (United States)

    van Ree, R; Hoffman, D R; van Dijk, W; Brodard, V; Mahieu, K; Koeleman, C A; Grande, M; van Leeuwen, W A; Aalberse, R C

    1995-05-01

    Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. We sought to characterize the allergen biochemically and immunologically. The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

  18. Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants.

    Science.gov (United States)

    Sunitha, Mellacheruvu; Srinath, Tamirisa; Reddy, Vudem Dashavantha; Rao, Khareedu Venkateswara

    2017-06-01

    Transgenic rice expressing pigeonpea Cc CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in CcCDR -transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes. Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants.

  19. Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickx, Antoni P.A.; Poor, Catherine B.; Jureller, Justin E.; Budzik, Jonathan M.; He, Chuan; Schneewind, Olaf (UC)

    2012-09-05

    Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA -- CNA{sub 1}, CNA{sub 2}, XNA and CNA{sub 3} -- each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA2 or CNA3 domains retain the ability to form pilus bundles. A mutant lacking the CNA{sub 1} isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp{sup 312}Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp{sup 312}Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA{sub 1} and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.

  20. Pregnancy-associated plasma protein-A (PAPP-A) and the proform of the eosinophil major basic protein (ProMBP) are associated with increased risk of death in heart failure patients

    DEFF Research Database (Denmark)

    Dembic, Maja; Hedley, Paula L.; Torp-Pedersen, Christian

    2017-01-01

    Risk stratification and patient management in heart failure (HF) is difficult due to the unpredictable progression of the disease, necessitating the development of reliable diagnostic biomarkers to facilitate decision-making in clinical practice. Pregnancy-associated plasma protein-A (PAPP...

  1. Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

    Science.gov (United States)

    Dos Santos, Fábio Neves; Tata, Alessandra; Belaz, Kátia Roberta Anacleto; Magalhães, Dilze Maria Argôlo; Luz, Edna Dora Martins Newman; Eberlin, Marcos Nogueira

    2017-03-01

    Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.

  2. TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits.

    Directory of Open Access Journals (Sweden)

    Dorothée Diogo

    Full Text Available Despite the success of genome-wide association studies (GWAS in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples, Exomechip genotyping (n = 18,409 case/control samples and targeted exon-sequencing (n = 2,236 case/controls samples to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2 independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3 x 10(-21, A928V (rs35018800, OR = 0.53, P = 1.2 x 10(-9, and I684S (rs12720356, OR = 0.86, P = 4.6 x 10(-7. Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6 x 10(-18, and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V may also protect against inflammatory bowel disease (IBD; P(omnibus = 0.005. Finally, in a phenome-wide association study (PheWAS assessing >500 phenotypes using electronic medical records (EMR in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.

  3. Prognostic value, clinical effectiveness and cost-effectiveness of high sensitivity C-reactive protein as a marker in primary prevention of major cardiac events.

    Science.gov (United States)

    Schnell-Inderst, Petra; Schwarzer, Ruth; Göhler, Alexander; Grandi, Norma; Grabein, Kristin; Stollenwerk, Björn; Klauß, Volker; Wasem, Jürgen; Siebert, Uwe

    2009-05-12

    In a substantial portion of patients (= 25%) with coronary heart disease (CHD), a myocardial infarction or sudden cardiac death without prior symptoms is the first manifestation of disease. The use of new risk predictors for CHD such as the high-sensitivity C-reactive Protein (hs-CRP) in addition to established risk factors could improve prediction of CHD. As a consequence of the altered risk assessment, modified preventive actions could reduce the number of cardiac death and non-fatal myocardial infarction. Does the additional information gained through the measurement of hs-CRP in asymptomatic patients lead to a clinically relevant improvement in risk prediction as compared to risk prediction based on traditional risk factors and is this cost-effective? A literature search of the electronic databases of the German Institute of Medical Documentation and Information (DIMDI) was conducted. Selection, data extraction, assessment of the study-quality and synthesis of information was conducted according to the methods of evidence-based medicine. Eight publications about predictive value, one publication on the clinical efficacy and three health-economic evaluations were included. In the seven study populations of the prediction studies, elevated CRP-levels were almost always associated with a higher risk of cardiovascular events and non-fatal myocardial infarctions or cardiac death and severe cardiovascular events. The effect estimates (odds ratio (OR), relative risk (RR), hazard ratio (HR)), once adjusted for traditional risk factors, demonstrated a moderate, independent association between hs-CRP and cardiac and cardiovascular events that fell in the range of 0.7 to 2.47. In six of the seven studies, a moderate increase in the area under the curve (AUC) could be detected by adding hs-CRP as a predictor to regression models in addition to established risk factors though in three cases this was not statistically significant. The difference [in the AUC] between the

  4. Prognostic value, clinical effectiveness and cost-effectiveness of high sensitivity C-reactive protein as a marker in primary prevention of major cardiac events

    Directory of Open Access Journals (Sweden)

    Klauß, Volker

    2009-05-01

    Full Text Available Background: In a substantial portion of patients (= 25% with coronary heart disease (CHD, a myocardial infarction or sudden cardiac death without prior symptoms is the first manifestation of disease. The use of new risk predictors for CHD such as the high-sensitivity C-reactive Protein (hs-CRP in addition to established risk factors could improve prediction of CHD. As a consequence of the altered risk assessment, modified preventive actions could reduce the number of cardiac death and non-fatal myocardial infarction. Research question: Does the additional information gained through the measurement of hs-CRP in asymptomatic patients lead to a clinically relevant improvement in risk prediction as compared to risk prediction based on traditional risk factors and is this cost-effective? Methods: A literature search of the electronic databases of the German Institute of Medical Documentation and Information (DIMDI was conducted. Selection, data extraction, assessment of the study-quality and synthesis of information was conducted according to the methods of evidence-based medicine. Results: Eight publications about predictive value, one publication on the clinical efficacy and three health-economic evaluations were included. In the seven study populations of the prediction studies, elevated CRP-levels were almost always associated with a higher risk of cardiovascular events and non-fatal myocardial infarctions or cardiac death and severe cardiovascular events. The effect estimates (odds ratio (OR, relative risk (RR, hazard ratio (HR, once adjusted for traditional risk factors, demonstrated a moderate, independent association between hs-CRP and cardiac and cardiovascular events that fell in the range of 0.7 to 2.47. In six of the seven studies, a moderate increase in the area under the curve (AUC could be detected by adding hs-CRP as a predictor to regression models in addition to established risk factors though in three cases this was not

  5. Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

    Science.gov (United States)

    Abbott, Jeffrey R; Palmer, Guy H; Howard, Chris J; Hope, Jayne C; Brown, Wendy C

    2004-12-01

    Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

  6. Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

    Science.gov (United States)

    De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

    2017-09-01

    Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably

  7. Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole

    International Nuclear Information System (INIS)

    Krizova, Hana; Zidek, Lukas; Stone, Martin J.; Novotny, Milos V.; Sklenar, Vladimir

    2004-01-01

    Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by 15 N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2-sec-4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature depencence of the motional parameters

  8. Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

    Science.gov (United States)

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Pandey, Awadh Bihari; Shivachandra, Sathish Bhadravati

    2017-05-01

    Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L ( 21 S to E 110 ; ∼30 kDa) and rH3L( 1 M to I 280 ; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 μg of each) was used for active immunization of adult mice (20μg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Fine mapping of a dominantly inherited powdery mildew resistance major-effect QTL, Pm1.1, in cucumber identifies a 41.1 kb region containing two tandemly arrayed cysteine-rich receptor-like protein kinase genes.

    Science.gov (United States)

    Xu, Xuewen; Yu, Ting; Xu, Ruixue; Shi, Yang; Lin, Xiaojian; Xu, Qiang; Qi, Xiaohua; Weng, Yiqun; Chen, Xuehao

    2016-03-01

    A dominantly inherited major-effect QTL for powdery mildew resistance in cucumber was fine mapped. Two tandemly arrayed cysteine-rich receptor-like protein kinase genes were identified as the most possible candidates. Powdery mildew (PM) is one of the most severe fungal diseases of cucumber (Cucumis sativus L.) and other cucurbit crops, but the molecular genetic mechanisms of powdery mildew resistance in cucurbits are still poorly understood. In this study, through marker-assisted backcrossing with an elite cucumber inbred line, D8 (PM susceptible), we developed a single-segment substitution line, SSSL0.7, carrying 95 kb fragment from PM resistance donor, Jin5-508, that was defined by two microsatellite markers, SSR16472 and SSR16881. A segregating population with 3600 F2 plants was developed from the SSSL0.7 × D8 mating; segregation analysis confirmed a dominantly inherited major-effect QTL, Pm1.1 in cucumber chromosome 1 underlying PM resistance in SSSL0.7. New molecular markers were developed through exploring the next generation resequenced genomes of Jin5-508 and D8. Linkage analysis and QTL mapping in a subset of the F2 plants delimited the Pm1.1 locus into a 41.1 kb region, in which eight genes were predicted. Comparative gene expression analysis revealed that two concatenated genes, Csa1M064780 and Csa1M064790 encoding the same function of a cysteine-rich receptor-like protein kinase, were the most likely candidate genes. GFP fusion protein-aided subcellular localization indicated that both candidate genes were located in the plasma membrane, but Csa1M064780 was also found in the nucleus. This is the first report of dominantly inherited PM resistance in cucumber. Results of this study will provide new insights into understanding the phenotypic and genetic mechanisms of PM resistance in cucumber. This work should also facilitate marker-assisted selection in cucumber breeding for PM resistance.

  10. Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses.

    Science.gov (United States)

    Khan, Shahneaz Ali; Waugh, Courtney; Rawlinson, Galit; Brumm, Jacqui; Nilsson, Karen; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2014-10-07

    Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  12. Proteomic analysis reveals a role for Bcl2-associated athanogene 3 and major vault protein in resistance to apoptosis in senescent cells by regulating ERK1/2 activation.

    Science.gov (United States)

    Pasillas, Martina P; Shields, Sarah; Reilly, Rebecca; Strnadel, Jan; Behl, Christian; Park, Robin; Yates, John R; Klemke, Richard; Gonias, Steven L; Coppinger, Judith A

    2015-01-01

    Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-03-15

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  14. Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation*

    Science.gov (United States)

    Pasillas, Martina P.; Shields, Sarah; Reilly, Rebecca; Strnadel, Jan; Behl, Christian; Park, Robin; Yates, John R.; Klemke, Richard; Gonias, Steven L.; Coppinger, Judith A.

    2015-01-01

    Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer. PMID:24997994

  15. Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

    Science.gov (United States)

    Sajó, Ráchel; Liliom, Károly; Muskotál, Adél; Klein, Agnes; Závodszky, Péter; Vonderviszt, Ferenc; Dobó, József

    2014-11-01

    Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

    Science.gov (United States)

    Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

    2013-04-01

    Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

  17. Double Strand Break Repair, one mechanism can hide another: Alternative non-homologous end joining

    International Nuclear Information System (INIS)

    Rass, E.; Grabarz, A.; Bertrand, P.; Lopez, B.S.

    2012-01-01

    DNA double strand breaks are major cytotoxic lesions encountered by the cells. They can be induced by ionizing radiation or endogenous stress and can lead to genetic instability. Two mechanisms compete for the repair of DNA double strand breaks: homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires DNA sequences homology and is initiated by single strand resection. Recently, advances have been made concerning the major steps and proteins involved in resection. NHEJ, in contrast, does not require sequence homology. The existence of a DNA double strand break repair mechanism, independent of KU and ligase IV, the key proteins of the canonical non homologous end joining pathway, has been revealed lately and named alternative non homologous end joining. The hallmarks of this highly mutagenic pathway are deletions at repair junctions and frequent use of distal micro-homologies. This mechanism is also initiated by a single strand resection of the break. The aim of this review is firstly to present recent data on single strand resection, and secondly the alternative NHEJ pathway, including a discussion on the fidelity of NHEJ. Based on current knowledge, canonical NHEJ does not appear as an intrinsically mutagenic mechanism, but in contrast, as a conservative one. The structure of broken DNA ends actually dictates the quality repair of the alternative NHEJ and seems the actual responsible for the mutagenesis attributed beforehand to the canonical NHEJ. The existence of this novel DNA double strand breaks repair mechanism needs to be taken into account in the development of radiosensitizing strategies in order to optimise the efficiency of radiotherapy. (authors)

  18. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  19. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    International Nuclear Information System (INIS)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-01-01

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  20. Augmented serum level of major histocompatibility complex class I-related chain A (MICA) protein and reduced NKG2D expression on NK and T cells in patients with cervical cancer and precursor lesions

    International Nuclear Information System (INIS)

    Arreygue-Garcia, Naela A; Delgado-Rizo, Vidal; Garcia-Iglesias, Trinidad; Hernandez-Flores, Georgina; Toro-Arreola, Susana del; Daneri-Navarro, Adrian; Toro-Arreola, Alicia del; Cid-Arregui, Angel; Gonzalez-Ramella, Oscar; Jave-Suarez, Luis F; Aguilar-Lemarroy, Adriana; Troyo-Sanroman, Rogelio; Bravo-Cuellar, Alejandro

    2008-01-01

    Cervical cancer is the second most common cancer in women worldwide. NK and cytotoxic T cells play an important role in the elimination of virus-infected and tumor cells through NKG2D activating receptors, which can promote the lysis of target cells by binding to the major histocompatibility complex class I-related chain A (MICA) proteins. Increased serum levels of MICA have been found in patients with epithelial tumors. The aim of this study was to compare the levels of soluble MICA (sMICA) and NKG2D-expressing NK and T cells in blood samples from patients with cervical cancer or precursor lesions with those from healthy donors. Peripheral blood with or without heparin was collected to obtain mononuclear cells or sera, respectively. Serum sMICA levels were measured by ELISA and NKG2D-expressing immune cells were analyzed by flow cytometry. Also, a correlation analysis was performed to associate sMICA levels with either NKG2D expression or with the stage of the lesion. Significant amounts of sMICA were detected in sera from nearly all patients. We found a decrease in the number of NKG2D-expressing NK and T cells in both cervical cancer and lesion groups when compared to healthy donors. Pearson analysis showed a negative correlation between sMICA and NKG2D-expressing T cells; however, we did not find a significant correlation when the analysis was applied to sMICA and NKG2D expression on NK cells. Our results show for the first time that high sMICA levels are found in sera from patients with both cervical cancer and precursor lesions when compared with healthy donors. We also observed a diminution in the number of NKG2D-expressing NK and T cells in the patient samples; however, a significant negative correlation between sMICA and NKG2D expression was only seen in T cells

  1. Compared with Raw Bovine Meat, Boiling but Not Grilling, Barbecuing, or Roasting Decreases Protein Digestibility without Any Major Consequences for Intestinal Mucosa in Rats, although the Daily Ingestion of Bovine Meat Induces Histologic Modifications in the Colon.

    Science.gov (United States)

    Oberli, Marion; Lan, Annaïg; Khodorova, Nadezda; Santé-Lhoutellier, Véronique; Walker, Francine; Piedcoq, Julien; Davila, Anne-Marie; Blachier, François; Tomé, Daniel; Fromentin, Gilles; Gaudichon, Claire

    2016-08-01

    Cooking may impair meat protein digestibility. When undigested proteins are fermented by the colon microbiota, they can generate compounds that potentially are harmful to the mucosa. This study addressed the effects of typical cooking processes and the amount of bovine meat intake on the quantity of undigested proteins entering the colon, as well as their effects on the intestinal mucosa. Male Wistar rats (n = 88) aged 8 wk were fed 11 different diets containing protein as 20% of energy. In 10 diets, bovine meat proteins represented 5% [low-meat diet (LMD)] or 15% [high-meat diet (HMD)] of energy, with the rest as total milk proteins. Meat was raw or cooked according to 4 processes (boiled, barbecued, grilled, or roasted). A meat-free diet contained only milk proteins. After 3 wk, rats ingested a (15)N-labeled meat meal and were killed 6 h later after receiving a (13)C-valine injection. Meat protein digestibility was determined from (15)N enrichments in intestinal contents. Cecal short- and branched-chain fatty acids and hydrogen sulfide were measured. Intestinal tissues were used for the assessment of protein synthesis rates, inflammation, and histopathology. Meat protein digestibility was lower in rats fed boiled meat (94.5% ± 0.281%) than in the other 4 groups (97.5% ± 0.0581%, P HMD) and on myeloperoxidase activity in the proximal colon (HMD > LMD), but not on other outcomes. The ingestion of bovine meat, whatever the cooking process and the intake amount, resulted in discrete histologic modifications of the colon (epithelium abrasion, excessive mucus secretion, and inflammation). Boiling bovine meat at a high temperature (100°C) for a long time (3 h) moderately lowered protein digestibility compared with raw meat and other cooking processes, but did not affect cecal bacterial metabolites related to protein fermentation. The daily ingestion of raw or cooked bovine meat had no marked effect on intestinal tissues, despite some slight histologic modifications

  2. Amino acids and proteins

    Science.gov (United States)

    A balanced, safe diet with proteins is important to meet nutritional requirements. Proteins occur in animal as well as vegetable products in important quantities. In some countries, many people obtain much of their protein from animal products. In other regions, the major portion of dietary protein ...

  3. Major Sport Venues

    Data.gov (United States)

    Department of Homeland Security — The Major Public Venues dataset is composed of facilities that host events for the National Association for Stock Car Auto Racing, Indy Racing League, Major League...

  4. Major Depression Among Adults

    Science.gov (United States)

    ... Depressive Episode Among Adolescents Data Sources Share Major Depression Definitions Major depression is one of the most ... Bethesda, MD 20892-9663 Follow Us Facebook Twitter YouTube Google Plus NIMH Newsletter NIMH RSS Feed NIMH ...

  5. Tacrolimus binding proteins 5 gene polymorphisms in major depressive disorder%重性抑郁障碍患者他克莫司结合蛋白5基因多态性分析

    Institute of Scientific and Technical Information of China (English)

    杨栋; 赵靖平; 喻妍; 吴仁容; 郭文斌

    2013-01-01

    Objective Tacrolimus binding proteins 5 (FKBP5) gene polymorphisms were analyzed in Chinese Han major depressive disorder (MDD) patients.Methods 263 major depressive disorder (MDD) patients who met the Diagnostic and Statistical Manual of Mental Disorders 4th Edition (DSM-Ⅳ)criterion were given antidepressant drugs for six weeks.The patients were divided into two groups of the responder group and the non-responder group,according to the decreasing rate of score of HAMD ≥ 50%and < 50% respectively.Genomic DNA was extracted from the blood samples,when patients being enrolled.Two polymorphisms (rs3800373 and rs1360780) of FKBP5 gene were genotyped using the ligase detection reaction.Results (1)The distribution frequencies of the SNP of rs3800373 showed no significant differences between responder group (GG + GT 45.7% ; TT 54.3%) and non-responder group (GG + GT47.6% ;T T 52.4%) (x2 =0.051,P > 0.05).The distribution frequencies of the SNP of rs1360780 showed no significant differences between responder group(CC 55.6% ; CT + TF 44.4%) and non-responder group (CC 51.2% ; CT + TT 48.8%) (x2 =0.256,P > 0.05).(2) No significant differences were found in rs3800373-rs1360780 haplotype frequencies between responder group and non-responder group (P > 0.05).(3) There were no significant differences in the incidence rate of total side effect and nausea adverse effect between the patients with different genotypes of rs3800373 and rs1360780 (P > 0.05).Conclusions There may be no significant differences in the genotype frequencies of FKBP5 gene rs3800373 and rs1360780 polymorphisms between responder group and non-responder group in Chinese Han MDD patients.%目的 分析中国汉族重性抑郁障碍患者他克莫司结合蛋白5(FKBP5)基因多态性.方法 对符合《美国诊断与统计手册精神障碍(第4版)》(DSM-Ⅳ)重性抑郁障碍诊断标准的263例患者予以单一抗抑郁药治疗,疗程6周.根据汉密尔顿

  6. Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

    Science.gov (United States)

    1990-05-01

    encoding the animals have shown that both cellular and humoral immune Sta58 protein antigen in E. coli. DNA sequence analysis of a responses occur after...infection, with the cellular immune 2.9-kilobase (kb) HindIl fragment carrying the Sta58 gene response being required for protection (16, 19, 25, 42...The first evidence of a 60-kDa common HtpB antigen) reacted strongly with protein antigens in the antigen family (Hsp6O) among procaryotes was based

  7. Neurobiology of Major Depressive Disorder

    Directory of Open Access Journals (Sweden)

    Rosa Villanueva

    2013-01-01

    Full Text Available We survey studies which relate abnormal neurogenesis to major depressive disorder. Clinically, descriptive gene and protein expression analysis and genetic and functional studies revised here show that individual alterations of a complex signaling network, which includes the hypothalamic-pituitary-adrenal axis; the production of neurotrophins and growth factors; the expression of miRNAs; the production of proinflammatory cytokines; and, even, the abnormal delivery of gastrointestinal signaling peptides, are able to induce major mood alterations. Furthermore, all of these factors modulate neurogenesis in brain regions involved in MDD, and are functionally interconnected in such a fashion that initial alteration in one of them results in abnormalities in the others. We highlight data of potential diagnostic significance and the relevance of this information to develop new therapeutic approaches. Controversial issues, such as whether neurogenesis is the basis of the disease or whether it is a response induced by antidepressant treatments, are also discussed.

  8. Wide cross-reactivity between Anopheles gambiae and Anopheles funestus SG6 salivary proteins supports exploitation of gSG6 as a marker of human exposure to major malaria vectors in tropical Africa

    Directory of Open Access Journals (Sweden)

    Petrarca Vincenzo

    2011-07-01

    Full Text Available Abstract Background The Anopheles gambiae gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector A. gambiae. However, malaria transmission in tropical Africa is sustained by three main vectors (A. gambiae, Anopheles arabiensis and Anopheles funestus and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the A. gambiae species complex whereas the A. funestus homologue, fSG6, is more divergent (80% identity with gSG6. The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6. Methods The A. funestus SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large A. gambiae prevalence (>75%. Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed. Results According to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level. Conclusions Comparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission

  9. Prospects after Major Trauma

    NARCIS (Netherlands)

    Holtslag, H.R.

    2007-01-01

    Introduction. After patients survived major trauma, their prospects, in terms of the consequences for functioning, are uncertain, which may impact severely on patient, family and society. The studies in this thesis describes the long-term outcomes of severe injured patients after major trauma. In

  10. A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

    Science.gov (United States)

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

    2013-07-08

    The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

  11. A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

    Directory of Open Access Journals (Sweden)

    Marco Ruggiero

    2013-07-01

    Full Text Available The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH(2D3, its two binding proteins that are the vitamin D receptor (VDR and the vitamin D-binding protein-derived macrophage activating factor (GcMAF. In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH(2D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

  12. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

    Science.gov (United States)

    Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

    2010-11-01

    Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

  13. Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency.

    Science.gov (United States)

    Saito, Shinta; Kurosawa, Aya; Adachi, Noritaka

    2016-08-01

    In successive reports from 2014 to 2015, X-ray repair cross-complementing protein 4 (XRCC4) has been identified as a novel causative gene of primordial dwarfism. XRCC4 is indispensable for non-homologous end joining (NHEJ), the major pathway for repairing DNA double-strand breaks. As NHEJ is essential for V(D)J recombination during lymphocyte development, it is generally believed that abnormalities in XRCC4 cause severe combined immunodeficiency. Contrary to expectations, however, no overt immunodeficiency has been observed in patients with primordial dwarfism harboring XRCC4 mutations. Here, we describe the various XRCC4 mutations that lead to disease and discuss their impact on NHEJ and V(D)J recombination.

  14. Major operations and activities

    Energy Technology Data Exchange (ETDEWEB)

    Black, D.G.

    1995-06-01

    This section of the 1994 Hanford Site Environmental Report summarizes the major operations and activities on the site. These operations and activities include site management, waste management, environmental restoration and corrective actions, and research and technology development.

  15. A major safety overhaul

    CERN Multimedia

    2003-01-01

    A redefined policy, a revamped safety course, an environmental project... the TIS (Technical Inspection and Safety) Division has begun a major safety overhaul. Its new head, Wolfgang Weingarten, explains to the Bulletin why and how this is happening.

  16. Allegheny County Major Rivers

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — This dataset contains locations of major rivers that flow through Allegheny County. These shapes have been taken from the Hydrology dataset. The Ohio River,...

  17. Major operations and activities

    International Nuclear Information System (INIS)

    Black, D.G.

    1995-01-01

    This section of the 1994 Hanford Site Environmental Report summarizes the major operations and activities on the site. These operations and activities include site management, waste management, environmental restoration and corrective actions, and research and technology development

  18. Functional Analysis of Homologous Recombination Repair Proteins HerA and NurA in the Thermophile Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong

    A number of DNA lesions are generated in each cell every day, among which double-stranded breaks (DSBs) constitute one of the most detrimental types of DNA damage. DSBs lead to genome instability, cell death, or even tumorigenesis in human, if not repaired timely. Two main pathways are known...... in the S/G2 phase of the cell cycle are preferentially repaired by HRR pathway, while NHEJ is the favorate pathway to repair DSBs in the G1 phase. Bacteria encode multiple pathways for DSB repair, including RecBCD, the primary HR pathway, SbcC-SbcD, and one backup system, RecFOR. In eukaryotes, the HRR...... pathway is mediated by Mre11-Rad50, homologs of bacterial SbcD-SbcC. However, numerous proteins and multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts, making many important mechanistic details poorly understood...

  19. Major international sport profiles.

    Science.gov (United States)

    Patel, Dilip R; Stier, Bernhard; Luckstead, Eugene F

    2002-08-01

    Sports are part of the sociocultural fabric of all countries. Although different sports have their origins in different countries, many sports are now played worldwide. International sporting events bring athletes of many cultures together and provide the opportunity not only for athletic competition but also for sociocultural exchange and understanding among people. This article reviews five major sports with international appeal and participation: cricket, martial arts, field hockey, soccer, and tennis. For each sport, the major aspects of physiological and biomechanical demands, injuries, and prevention strategies are reviewed.

  20. Isolamento da globulina majoritária, digestibilidade in vivo e in vitro das proteínas do tremoço-doce (Lupinus albus L., var. Multolupa Isolation of major globulin, in vivo and in vitro digestibility of proteins from sweet lupin (Lupinus albus L. var. Multolupa

    Directory of Open Access Journals (Sweden)

    Valdir Augusto Neves

    2006-12-01

    Full Text Available Os objetivos do trabalho foram isolar, purificar e estudar algumas propriedades da fração globulina majoritária de tremoço-doce, var. Multolupa; assim como avaliar as características de digestibilidade da farinha e frações isoladas. As frações protéicas foram separadas por fracionamento diferencial com uso de diferentes solventes. A globulina majoritária de tremoço-doce foi isolada, purificada por cromatografia em Q-Sepharose, revelando um único pico de proteína. Apresentou um peso molecular de 162,5 ± 10,0 kDa, determinado por cromatografia em Sephacryl S-300, e subunidades entre 20-70 kDa em PAGE-SDS. A solubilidade em função do pH e concentrações de NaCl revelaram curva típica dessa fração. A digestibilidade da proteína da farinha e das frações albumina, globulina e glutelina foi avaliada por experimentos in vitro e in vivo e se revelou alta para a fração globulina, seguida pela glutelina, albumina e farinha. A digestibilidade in vivo da fração globulina, tanto aparente quanto verdadeira, não diferiu significativamente daquela determinada para a caseína. Apesar da alta digestibilidade da fração majoritária e frações protéicas, a utilização como única fonte de proteína nas dietas revelou valores baixos de RPLR (razão protéica líquida relativa, indicando ser insuficiente para sustentar o crescimento dos animais, comparativamente à caseína.The aim of the present work was to isolate, purify and study some properties of the major globulin fraction from sweet lupin, var. Multolupa; as well as to evaluate the digestibility features of flour and isolated fractions. The protein fractions were separated by differential fractionation with different solvents. The lupin major globulin was isolated, purified by Q-Sepharose chromatography when it showed only one protein peak. The major protein presented a molecular weight of 162.5 ± 10.0 kDa, determined on Sephacryl S-300 chromatography; and subunits between 20

  1. Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

    Science.gov (United States)

    Caruso, Paola; Dunmore, Benjamin J; Schlosser, Kenny; Schoors, Sandra; Dos Santos, Claudia; Perez-Iratxeta, Carol; Lavoie, Jessie R; Zhang, Hui; Long, Lu; Flockton, Amanda R; Frid, Maria G; Upton, Paul D; D'Alessandro, Angelo; Hadinnapola, Charaka; Kiskin, Fedir N; Taha, Mohamad; Hurst, Liam A; Ormiston, Mark L; Hata, Akiko; Stenmark, Kurt R; Carmeliet, Peter; Stewart, Duncan J; Morrell, Nicholas W

    2017-12-19

    Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1 , and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Pulmonary vascular and

  2. Major New Initiatives

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Major New Initiatives. Multi-party multi-rate video conferencing OOPS. Live Lecture OOPS. Rural ATM Machine Vortex. Finger print detection HP-IITM. Medical Diagnostic kit NeuroSynaptic. LCD projection system TeNeT. Web Terminal MeTeL Midas. Entertainment ...

  3. Major planning enquiries

    Energy Technology Data Exchange (ETDEWEB)

    Shore, P

    1978-11-01

    This is a speech delivered by the U.K. Secretary of State for the Environment in Manchester (UK) on September 13th 1978. It outlines the Minister's views on the role and significance of major planning inquiries - such as that proposed to be held on the Commercial Demonstration Fast Reactor. (CDFR) (author).

  4. Major Biomass Conference

    Science.gov (United States)

    Top Scientists, Industry and Government Leaders to Gather for Major Biomass Conference America, South America and Europe will focus on building a sustainable, profitable biomass business at the Third Biomass Conference of the Americas in Montreal. Scheduled presentations will cover all biomass

  5. Unity in Major Themes

    DEFF Research Database (Denmark)

    Booss-Bavnbek, Bernhelm; Davis, Philip J.

    We describe and explain the desire, common among mathematicians, both for unity and independence in its major themes. In the dialogue that follows, we express our spontaneous and considered judgment and reservations; by contrasting the development of mathematics as a goal-driven process as opposed...

  6. The nucleoid-associated proteins H-NS and FIS modulate the DNA supercoiling response of the pel genes, the major virulence factors in the plant pathogen bacterium Dickeya dadantii

    Science.gov (United States)

    Ouafa, Zghidi-Abouzid; Reverchon, Sylvie; Lautier, Thomas; Muskhelishvili, Georgi; Nasser, William

    2012-01-01

    Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Previously we found that the pel gene expression is modulated by H-NS and FIS, two nucleoid-associated proteins (NAPs) modulating the DNA topology. Here, we show that relaxation of the DNA in growing D. dadantii cells decreases the expression of pel genes. Deletion of fis aggravates, whereas that of hns alleviates the negative impact of DNA relaxation on pel expression. We further show that H-NS and FIS directly bind the pelE promoter and that the response of D. dadantii pel genes to stresses that induce DNA relaxation is modulated, although to different extents, by H-NS and FIS. We infer that FIS acts as a repressor buffering the negative impact of DNA relaxation on pel gene transcription, whereas H-NS fine-tunes the response of virulence genes precluding their expression under suboptimal conditions of supercoiling. This novel dependence of H-NS effect on DNA topology expands our understanding of the role of NAPs in regulating the global bacterial gene expression and bacterial pathogenicity. PMID:22275524

  7. The major-effect quantitative trait locus CsARN6.1 encodes an AAA ATPase domain-containing protein that is associated with waterlogging stress tolerance by promoting adventitious root formation.

    Science.gov (United States)

    Xu, Xuewen; Ji, Jing; Xu, Qiang; Qi, Xiaohua; Weng, Yiqun; Chen, Xuehao

    2018-03-01

    In plants, the formation of hypocotyl-derived adventitious roots (ARs) is an important morphological acclimation to waterlogging stress; however, its genetic basis remains fragmentary. Here, through combined use of bulked segregant analysis-based whole-genome sequencing, SNP haplotyping and fine genetic mapping, we identified a candidate gene for a major-effect QTL, ARN6.1, that was responsible for waterlogging tolerance due to increased AR formation in the cucumber line Zaoer-N. Through multiple lines of evidence, we show that CsARN6.1 is the most possible candidate for ARN6.1 which encodes an AAA ATPase. The increased formation of ARs under waterlogging in Zaoer-N could be attributed to a non-synonymous SNP in the coiled-coil domain region of this gene. CsARN6.1 increases the number of ARs via its ATPase activity. Ectopic expression of CsARN6.1 in Arabidopsis resulted in better rooting ability and lateral root development in transgenic plants. Transgenic cucumber expressing the CsARN6.1 Asp allele from Zaoer-N exhibited a significant increase in number of ARs compared with the wild type expressing the allele from Pepino under waterlogging conditions. Taken together, these data support that the AAA ATPase gene CsARN6.1 has an important role in increasing cucumber AR formation and waterlogging tolerance. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  8. The Ursa Major supercluster

    International Nuclear Information System (INIS)

    Schuch, N.J.

    1983-01-01

    An optical and a radio survey have been carried out. The optical observations consist of a spectroscopic survey in which redshift data for cluster galaxies and optical identifications of radio sources were obtained with the 98-inch Isaac Newton telescope at the Royal Greenwich Observatory, and the 200-inch Hale telescope; the photographic survey in B, V and R colors was made with the 48-inch Schmidt telescope at Palomar. Some results on the galaxy distribution in the Ursa Major supercluster are briefly discussed. (Auth.)

  9. Securing Major Events

    International Nuclear Information System (INIS)

    Loeoef, Susanna

    2013-01-01

    When asked why the IAEA should provide nuclear security support to countries that organize large public events, Nuclear Security Officer Sophia Miaw answers quickly and without hesitation. ''Imagine any major public event such as the Olympics, a football championship, or an Expo. If a dirty bomb were to be exploded at a site where tens of thousands of people congregate, the radioactive contamination would worsen the effects of the bomb, increase the number of casualties, impede a rapid emergency response, and cause long term disruption in the vicinity,'' she said. Avoiding such nightmarish scenarios is the driving purpose behind the assistance the IAEA offers States that host major sporting or other public events. The support can range from a single training course to a comprehensive programme that includes threat assessment, training, loaned equipment and exercises. The type and scope of assistance depends on the host country's needs. ''We incorporate nuclear security measures into their security plan. We don't create anything new,'' Miaw said

  10. Major Depressive Disorder

    Directory of Open Access Journals (Sweden)

    G Grobler

    2013-08-01

    Full Text Available The treatment guideline draws on several international guidelines: (iPractice Guidelines of the American Psychiatric Association (APAfor the Treatment of Patients with Major Depressive Disorder, SecondEdition;[1](ii Clinical Guidelines for the Treatment of DepressiveDisorders by the Canadian Psychiatric Association and the CanadianNetwork for Mood and Anxiety Treatments (CANMAT;[2](iiiNational Institute for Clinical Excellence (NICE guidelines;[3](iv RoyalAustralian and New Zealand College of Psychiatrists Clinical PracticeGuidelines Team for Depression (RANZCAP;[4](v Texas MedicationAlgorithm Project (TMAP Guidelines;[5](vi World Federation ofSocieties of Biological Psychiatry (WFSBP Treatment Guideline forUnipolar Depressive Disorder;[6]and (vii British Association forPsychopharmacology Guidelines.[7

  11. DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Hennequin, C.; Averbeck, D.

    1999-01-01

    Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

  12. The advertisement role of major urinary proteins in mice

    Czech Academy of Sciences Publication Activity Database

    Stopka, Pavel; Janotová, K.; Heyrovský, D.

    2007-01-01

    Roč. 91, - (2007), s. 667-670 ISSN 0031-9384 Grant - others:GA ČR(CZ) GA206/04/0493 Institutional research plan: CEZ:AV0Z50450515 Keywords : oestrus * oestrous cycle * MUP Subject RIV: EG - Zoology Impact factor: 2.561, year: 2007

  13. Synthesis of the major storage protein, hordein, in barley

    DEFF Research Database (Denmark)

    Giese, Nanna Henriette; Andersen, B.; Doll, Hans

    1983-01-01

    A liquid culture system for culturing detached spikes of barley (Hordeum vulgare L.) at different nutritional levels was established. The synthesis of hordein polypeptides was studied by pulse-labeling with [14C]sucrose at different stages of development and nitrogen (N) nutrition. All polypeptides...

  14. Identification of the major allergens of Indian scad (Decapterus russelli).

    Science.gov (United States)

    Misnan, Rosmilah; Murad, Shahnaz; Jones, Meinir; Taylor, Graham; Rahman, Dinah; Arip, Masita; Abdullah, Noormalin; Mohamed, Jamaluddin

    2008-12-01

    The purpose of this study was to characterize major allergens of Indian scad (Decapterus russelli) which is among the most commonly consumed fish in Malaysia. Raw and cooked extracts of the fish were prepared. Protein profiles and IgE binding patterns were produced by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from subjects with fish allergy. The major allergens of the fish were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry of the peptide digests. The SDS-PAGE of the raw extract revealed 27 protein fractions over a wide molecular weight range, while the cooked extract demonstrated only six protein fractions. The 1-DE immunoblotting detected 14 IgE-binding proteins, with a molecular weight range from 90 to fish. The approximately 12 kDa band was a heat-resistant protein while the approximately 51 and 46 kDa proteins were sensitive to heat. The 2-DE gel profile of the raw extract demonstrated > 100 distinct protein spots and immunoblotting detected at least 10 different major IgE reactive spots with molecular masses as expected and isoelectric point (pI) values ranging from 4.0 to 7.0. A comparison of the major allergenic spot sequences of the 12 kDa proteins with known protein sequences in databases revealed extensive similarity with fish parvalbumin. In conclusion, this study demonstrated that a parvalbumin which is similar to Gad c 1 is the major allergen of Indian scad. Interestingly, we also detected heat-sensitive proteins as major allergenic components in our fish allergy patients.

  15. Modeling Non-homologous End Joining

    Science.gov (United States)

    Li, Yongfeng

    2013-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed

  16. Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks.

    Science.gov (United States)

    Stephanou, Nicolas C; Gao, Feng; Bongiorno, Paola; Ehrt, Sabine; Schnappinger, Dirk; Shuman, Stewart; Glickman, Michael S

    2007-07-01

    Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

  17. Proteins: Chemistry, Characterization, and Quality

    NARCIS (Netherlands)

    Sforza, S.; Tedeschi, T.; Wierenga, P.A.

    2016-01-01

    Proteins are one of the major macronutrients in food, and several traditional food commodities are good sources of proteins (meat, egg, milk and dairy products, fish, and soya). Proteins are polymers made by 20 different amino acids. They might undergo desired or undesired chemical or enzymatic

  18. Weaving History through the Major

    Science.gov (United States)

    Mayfield, Betty

    2014-01-01

    The benefits of including the study of the history of mathematics in the education of mathematics majors have been discussed at length elsewhere. Many colleges and universities now offer a History of Mathematics course for mathematics majors, for mathematics education majors, or for general credit. At Hood College, we emphasize our commitment to…

  19. Do Biology Majors Really Differ from Non–STEM Majors?

    Science.gov (United States)

    Cotner, Sehoya; Thompson, Seth; Wright, Robin

    2017-01-01

    Recent calls to action urge sweeping reform in science education, advocating for improved learning for all students—including those majoring in fields beyond the sciences. However, little work has been done to characterize the differences—if any exist—between students planning a career in science and those studying other disciplines. We describe an attempt to clarify, in broad terms, how non–STEM (science, technology, engineering, and mathematics) majors differ from life sciences majors, and how they are similar. Using survey responses and institutional data, we find that non–STEM majors are not unilaterally science averse; non–STEM majors are more likely than biology majors to hold misconceptions about the nature of science, yet they are not completely ignorant of how science works; non–STEM majors are less likely than biology majors to see science as personally relevant; and non–STEM majors populations are likely to be more diverse—with respect to incoming knowledge, perceptions, backgrounds, and skills—than a biology majors population. We encourage science educators to consider these characteristics when designing curricula for future scientists or simply for a well-informed citizenry. PMID:28798210

  20. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  1. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  2. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  3. Interdisciplinary Project Experiences: Collaboration between Majors and Non-Majors

    Science.gov (United States)

    Smarkusky, Debra L.; Toman, Sharon A.

    2014-01-01

    Students in computer science and information technology should be engaged in solving real-world problems received from government and industry as well as those that expose them to various areas of application. In this paper, we discuss interdisciplinary project experiences between majors and non-majors that offered a creative and innovative…

  4. Do Biology Majors Really Differ from Non-STEM Majors?

    Science.gov (United States)

    Cotner, Sehoya; Thompson, Seth; Wright, Robin

    2017-01-01

    Recent calls to action urge sweeping reform in science education, advocating for improved learning for all students-including those majoring in fields beyond the sciences. However, little work has been done to characterize the differences-if any exist-between students planning a career in science and those studying other disciplines. We describe an attempt to clarify, in broad terms, how non-STEM (science, technology, engineering, and mathematics) majors differ from life sciences majors, and how they are similar. Using survey responses and institutional data, we find that non-STEM majors are not unilaterally science averse; non-STEM majors are more likely than biology majors to hold misconceptions about the nature of science, yet they are not completely ignorant of how science works; non-STEM majors are less likely than biology majors to see science as personally relevant; and non-STEM majors populations are likely to be more diverse-with respect to incoming knowledge, perceptions, backgrounds, and skills-than a biology majors population. We encourage science educators to consider these characteristics when designing curricula for future scientists or simply for a well-informed citizenry. © 2017 S. Cotner et al. CBE—Life Sciences Education © 2017 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). 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).

  5. COMPOSITIONAL CHANGES OF MAJOR CHEMICAL ...

    African Journals Online (AJOL)

    Preferred Customer

    3Ege University, Department of Botany, Bornova, Izmir, Turkey ... received from seed germ [6], which is used as alternative protein source for the industry. The ... and sugars of carob pods during fruit development were studied as well.

  6. Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

    Science.gov (United States)

    Moscariello, Mario; Wieloch, Radi; Kurosawa, Aya; Li, Fanghua; Adachi, Noritaka; Mladenov, Emil; Iliakis, George

    2015-07-01

    Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Unconventional Internships for English Majors.

    Science.gov (United States)

    Otto, Don H.

    After five years of research, the English department at St. Cloud (Minnesota) State University created an internship program for English majors. The philosophy behind the program is that the typical experience of the English major in college is excellent preparation for what the college graduate will be doing in most careers in business,…

  8. Do You Have Major Depression?

    Science.gov (United States)

    ... of this page please turn Javascript on. Feature: Depression Do You Have Major Depression? Past Issues / Fall 2009 Table of Contents Simple ... member may have major depression. —NIMH Types of Depression Just like other illnesses, such as heart disease, ...

  9. Dairy Proteins and Energy Balance

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist

    High protein diets affect energy balance beneficially through decreased hunger, enhanced satiety and increased energy expenditure. Dairy products are a major source of protein. Dairy proteins are comprised of two classes, casein (80%) and whey proteins (20%), which are both of high quality......, but casein is absorbed slowly and whey is absorbed rapidly. The present PhD study investigated the effects of total dairy proteins, whey, and casein, on energy balance and the mechanisms behind any differences in the effects of the specific proteins. The results do not support the hypothesis that dairy...... proteins, whey or casein are more beneficial than other protein sources in the regulation of energy balance, and suggest that dairy proteins, whey or casein seem to play only a minor role, if any, in the prevention and treatment of obesity....

  10. The Pentapeptide Repeat Proteins

    OpenAIRE

    Vetting, Matthew W.; Hegde, Subray S.; Fajardo, J. Eduardo; Fiser, Andras; Roderick, Steven L.; Takiff, Howard E.; Blanchard, John S.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F]-[S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Myc...

  11. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...

  12. Protein annotation from protein interaction networks and Gene Ontology.

    Science.gov (United States)

    Nguyen, Cao D; Gardiner, Katheleen J; Cios, Krzysztof J

    2011-10-01

    We introduce a novel method for annotating protein function that combines Naïve Bayes and association rules, and takes advantage of the underlying topology in protein interaction networks and the structure of graphs in the Gene Ontology. We apply our method to proteins from the Human Protein Reference Database (HPRD) and show that, in comparison with other approaches, it predicts protein functions with significantly higher recall with no loss of precision. Specifically, it achieves 51% precision and 60% recall versus 45% and 26% for Majority and 24% and 61% for χ²-statistics, respectively. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Stenting of major airway constriction

    International Nuclear Information System (INIS)

    Wu Xiaomei

    2002-01-01

    Objective: To investigate the correlated issues in the stenting treatment of major airway constriction. Methods: Nineteen cases of major airway stenting procedure were studied retrospectively. The clinical choice of stents of different advantages or deficiencies were discussed. The importance of intravenous anesthesia supporting, life-parameters monitoring during the procedures and the prevention of complications were analysed. Results: Under intravenous and local anesthesia, 19 Wallstents had been successively placed and relieved 19 cases of major airway constrictions due to malignant or benign diseases (15 of tumors, 3 of tuberculosis, 1 of tracheomalacia). Intravenous anesthesia and life-parameters monitoring had made the procedures more safe and precise. Conclusions: Major airway stenting is an reliable method for relieving tracheobronchial stenosis; and intravenous anesthesia supporting and life-parameters monitoring guarantee the satisfactions of procedures

  14. Major hazards onshore and offshore

    International Nuclear Information System (INIS)

    1992-01-01

    This symposium continues the tradition of bringing together papers on a topic of current interest and importance in terms of process safety - in this case, Major Hazards Onshore and Offshore. Lord Cullen in his report on the Piper Alpha disaster has, in effect, suggested that the experience gained in the control of major hazards onshore during the 1980s should be applied to improve safety offshore during the 1990s. This major three-day symposium reviews what has been learned so far with regard to major hazards and considers its present and future applications both onshore and offshore. The topics covered in the programme are wide ranging and deal with all aspects of legislation, the application of regulations, techniques for evaluating hazards and prescribing safety measures in design, construction and operation, the importance of the human factors, and recent technical developments in protective measures, relief venting and predicting the consequences of fires and explosions. (author)

  15. Liquid in the major incision

    International Nuclear Information System (INIS)

    Herrera Jaramillo, Diego Alberto; Ortega Jaramillo, Hector

    2003-01-01

    We present the case of a patient with spill pleural extending in the left major incision. In the chest thorax PA, we could observe one of the complex radiographic appearances that take the reconfiguration of fluid in this localization, being this appearance dependent of the patient's position. Some points are also discussed on the anatomy of the major incisions and some of their radiographic characteristics

  16. Pathways for double-strand break repair in genetically unstable Z-DNA-forming sequences.

    Science.gov (United States)

    Kha, Diem T; Wang, Guliang; Natrajan, Nithya; Harrison, Lynn; Vasquez, Karen M

    2010-05-14

    DNA can adopt many structures that differ from the canonical B-form, and several of these non-canonical DNA structures have been implicated in genetic instability associated with human disease. Earlier, we found that Z-DNA causes DNA double-strand breaks (DSBs) in mammalian cells that can result in large-scale deletions and rearrangements. In contrast, the same Z-DNA-forming CG repeat in Escherichia coli resulted in only small contractions or expansions within the repeat. This difference in the Z-DNA-induced mutation spectrum between mammals and bacteria might be due to different mechanisms for DSB repair; in mammalian cells, non-homologous end-joining (NHEJ) is a major DSB repair pathway, while E. coli do not contain this system and typically use homologous recombination (HR) to process DSBs. To test the extent to which the different DSB repair pathways influenced the Z-DNA-induced mutagenesis, we engineered bacterial E.coli strains to express an inducible NHEJ system, to mimic the situation in mammalian cells. Mycobacterium tuberculosis NHEJ proteins Ku and ligase D (LigD) were expressed in E.coli cells in the presence or absence of HR, and the Z-DNA-induced mutations were characterized. We found that the presence of the NHEJ mechanism markedly shifted the mutation spectrum from small deletions/insertions to large-scale deletions (from 2% to 24%). Our results demonstrate that NHEJ plays a role in the generation of Z-DNA-induced large-scale deletions, suggesting that this pathway is associated with DNA structure-induced destabilization of genomes from prokaryotes to eukaryotes. (c) 2010 Elsevier Ltd. All rights reserved.

  17. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  18. Synthesis of Lipidated Proteins.

    Science.gov (United States)

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

  19. Major Decisions: Motivations for Selecting a Major, Satisfaction, and Belonging

    Science.gov (United States)

    Soria, Krista M.; Stebleton, Michael

    2013-01-01

    In this paper, we analyzed the relationship between students' motivations for choosing academic majors and their satisfaction and sense of belonging on campus. Based on a multi-institutional survey of students who attended large, public, research universities in 2009, the results suggest that external extrinsic motivations for selecting a major…

  20. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  1. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  2. Whey Protein

    Science.gov (United States)

    ... reliable information about the safety of taking whey protein if you are pregnant or breast feeding. Stay on the safe side and avoid use. Milk allergy: If you are allergic to cow's milk, avoid using whey protein.

  3. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

    DEFF Research Database (Denmark)

    Lundgren, J D; Davey, R T; Lundgren, B

    1991-01-01

    Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants...

  4. Personality, academic majors and performance

    DEFF Research Database (Denmark)

    Vedel, Anna; Thomsen, Dorthe Kirkegaard; Larsen, Lars

    2015-01-01

    Personality–performance research typically uses samples of psychology students without questioning their representativeness. The present article reports two studies challenging this practice. Study 1: group differences in the Big Five personality traits were explored between students (N = 1067......) in different academic majors (medicine, psychology, law, economics, political science, science, and arts/humanities), who were tested immediately after university enrolment. Study 2: six and a half years later the students’ academic records were obtained, and predictive validity of the Big Five personality...... traits and their subordinate facets was examined in the various academic majors in relation to Grade Point Average (GPA). Significant group differences in all Big Five personality traits were found between students in different academic majors. Also, variability in predictive validity of the Big Five...

  5. Major disruption process in tokamak

    International Nuclear Information System (INIS)

    Kurita, Gen-ichi; Azumi, Masafumi; Tuda, Takashi; Takizuka, Tomonori; Tsunematsu, Toshihide; Tokuda, Shinji; Itoh, Kimitaka; Takeda, Tatsuoki

    1981-11-01

    The major disruption in a cylindrical tokamak is investigated by using the multi-helicity code, and the destabilization of the 3/2 mode by the mode coupling with the 2/1 mode is confirmed. The evolution of the magnetic field topology caused by the major disruption is studied in detail. The effect of the internal disruption on the 2/1 magnetic island width is also studied. The 2/1 magnetic island is not enhanced by the flattening of the q-profile due to the internal disruption. (author)

  6. Major KEEP Findings, 1971 - 1975.

    Science.gov (United States)

    Kamehameha Schools, Honolulu, HI. Kamehameha Early Education Project.

    This report lists the 34 major research findings from the Kamehameha Early Education Program (KEEP) for the years 1971-1975. Each finding is accompanied by a listing of KEEP technical reports and working papers which contain information relevant to that finding. Included among areas covered in the findings are: (1) student motivation, (2) teacher…

  7. Dynamic range majority data structures

    DEFF Research Database (Denmark)

    Elmasry, Amr Ahmed Abd Elmoneim; He, Meng; Munro, J. Ian

    2011-01-01

    Given a set P of n coloured points on the real line, we study the problem of answering range α-majority (or "heavy hitter") queries on P. More specifically, for a query range Q, we want to return each colour that is assigned to more than an α-fraction of the points contained in Q. We present a ne...

  8. Understanding Business Majors' Learning Styles

    Science.gov (United States)

    Giordano, James; Rochford, Regina A.

    2005-01-01

    Recently, business education programs have experienced a decline in enrollment and an increase in attrition. To understand these issues and recommend solutions, the learning styles of 503 first-year business majors at an urban community college were examined. The results demonstrated that: (a) 94% of the participants were analytic learners; (b)…

  9. Epidemiology of major depressive disorder

    NARCIS (Netherlands)

    Stegenga, B.T.

    2011-01-01

    Major depressive disorder (MDD) is a serious health problem and will be the second leading cause of burden of disease worldwide by 2030. To be able to prevent MDD, insight into risk factors for the onset of MDD is of clear importance. On the other hand, if onset of MDD has occurred, one may argue

  10. Rediscovering Major N. Clark Smith.

    Science.gov (United States)

    Buckner, Reginald T.

    1985-01-01

    Historians of American music education have yet to recognize a Black music educator as important and worthy of observation. This article discusses a candidate--Major Nathaniel Clark Smith, a little-known Black music educator, composer of more than a hundred works, businessman, humanitarian, and teacher of numerous big-name jazz musicians. (RM)

  11. Endocrinopathies in thalassemia major patient

    Science.gov (United States)

    Lubis, D. A.; Yunir, E. M.

    2018-03-01

    Advanced in chelation therapy and regular blood transfusion have marked improvements in the life expectancy of patients with thalassemia major, however these patients still have to deal with several complications. We report a 19-year-old male, presented with multiple endocrine complication-related thalassemia; hypogonadism, short stature, osteoporosis with history of fracture, and subclinical hypothyroid.

  12. Physics momentum 'stars' draw majors

    CERN Multimedia

    Lindström, I

    2003-01-01

    Over the past decade, the number of University of Arizona students declaring physics as their major has doubled, amid a national decline. According to a recent report by the National Task Force on Undergraduate Physics, it is the university's dedication to its undergraduate physics program which draws students in (1 page).

  13. Major Depression Can Be Prevented

    Science.gov (United States)

    Munoz, Ricardo F.; Beardslee, William R.; Leykin, Yan

    2012-01-01

    The 2009 Institute of Medicine report on prevention of mental, emotional, and behavioral disorders (National Research Council & Institute of Medicine, 2009b) presented evidence that major depression can be prevented. In this article, we highlight the implications of the report for public policy and research. Randomized controlled trials have shown…

  14. Dirichlet polynomials, majorization, and trumping

    International Nuclear Information System (INIS)

    Pereira, Rajesh; Plosker, Sarah

    2013-01-01

    Majorization and trumping are two partial orders which have proved useful in quantum information theory. We show some relations between these two partial orders and generalized Dirichlet polynomials, Mellin transforms, and completely monotone functions. These relations are used to prove a succinct generalization of Turgut’s characterization of trumping. (paper)

  15. Managemant of NASA's major projects

    Science.gov (United States)

    James, L. B.

    1973-01-01

    Approaches used to manage major projects are studied and the existing documents on NASA management are reviewed. The work consists of: (1) the project manager's role, (2) request for proposal, (3) project plan, (4) management information system, (5) project organizational thinking, (6) management disciplines, (7) important decisions, and (8) low cost approach.

  16. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  17. Recent advances in racemic protein crystallography.

    Science.gov (United States)

    Yan, Bingjia; Ye, Linzhi; Xu, Weiliang; Liu, Lei

    2017-09-15

    Solution of the three-dimensional structures of proteins is a critical step in deciphering the molecular mechanisms of their bioactivities. Among the many approaches for obtaining protein crystals, racemic protein crystallography has been developed as a unique method to solve the structures of an increasing number of proteins. Exploiting unnatural protein enantiomers in crystallization and resolution, racemic protein crystallography manifests two major advantages that are 1) to increase the success rate of protein crystallization, and 2) to obviate the phase problem in X-ray diffraction. The requirement of unnatural protein enantiomers in racemic protein crystallography necessitates chemical protein synthesis, which is hitherto accomplished through solid phase peptide synthesis and chemical ligation reactions. This review highlights the fundamental ideas of racemic protein crystallography and surveys the harvests in the field of racemic protein crystallography over the last five years from early 2012 to late 2016. Copyright © 2017. Published by Elsevier Ltd.

  18. Characterisation of a major enzyme of bovine nitrogen metabolism

    CSIR Research Space (South Africa)

    Mathomu, LM

    2010-09-01

    Full Text Available of cellular protein metabolism (Curthoys & Watford, 1995; Meister, 1974). Glutamine functions as a major inter-organ transport form of nitrogen, carbon and serves as a source of energy between tissues such as brain, liver, kidney and even muscles...

  19. The interface of protein structure, protein biophysics, and molecular evolution

    Science.gov (United States)

    Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

    2012-01-01

    Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

  20. Distance majorization and its applications.

    Science.gov (United States)

    Chi, Eric C; Zhou, Hua; Lange, Kenneth

    2014-08-01

    The problem of minimizing a continuously differentiable convex function over an intersection of closed convex sets is ubiquitous in applied mathematics. It is particularly interesting when it is easy to project onto each separate set, but nontrivial to project onto their intersection. Algorithms based on Newton's method such as the interior point method are viable for small to medium-scale problems. However, modern applications in statistics, engineering, and machine learning are posing problems with potentially tens of thousands of parameters or more. We revisit this convex programming problem and propose an algorithm that scales well with dimensionality. Our proposal is an instance of a sequential unconstrained minimization technique and revolves around three ideas: the majorization-minimization principle, the classical penalty method for constrained optimization, and quasi-Newton acceleration of fixed-point algorithms. The performance of our distance majorization algorithms is illustrated in several applications.

  1. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis.

    Science.gov (United States)

    Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S

    2014-10-01

    Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Protein degradation and protection against misfolded or damaged proteins

    Science.gov (United States)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  3. Protein stability: a crystallographer’s perspective

    Science.gov (United States)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  4. Epidemiology of major depressive disorder

    OpenAIRE

    Stegenga, B.T.

    2011-01-01

    Major depressive disorder (MDD) is a serious health problem and will be the second leading cause of burden of disease worldwide by 2030. To be able to prevent MDD, insight into risk factors for the onset of MDD is of clear importance. On the other hand, if onset of MDD has occurred, one may argue that different course patterns of MDD can be identified and that it is essential to examine their relationship to symptoms and function over time. Insight into these course patterns could assist in p...

  5. Aostra claims major oilsands breakthrough

    International Nuclear Information System (INIS)

    Anon.

    1991-01-01

    This paper reports that Alberta Oil Sands Technology and Research Authority (Aostra) has completed a horizontal well in-situ steam injection project it calls a major breakthrough in commercially producing bitumen from the bast Athabasca oilsands deposit in Alberta. Aostra the its $71 million (Canadian) proof of concept pilot underground test facility (UTF) near Fort McMurray, achieved a 60% bitumen recovery rate, compared with less than 20% recovery typically achieved with Alberta bitumen. More than 100,000 bbl of bitumen was produced during the project

  6. Vanpooling: the three major approaches

    Energy Technology Data Exchange (ETDEWEB)

    Sears, P.M.

    1979-08-01

    The manual provides technical assistance to existing or prospective vanpool sponsors. It is designed to help them promote vanpooling in its three major approaches: employer sponsored, third party sponsored, and driver owned and operated. The first chapter is an overview of vanpooling and a second chapter, on vanpool marketing, is addressed to ridesharing coordinators and others whose responsibilities include the promotion of vanpooling. Some fact sheets on the three approaches provide convenient summaries of the needs and opportunities of each approach and suggest solutions to practical problems likely to be encountered in starting new vanpool programs.

  7. Majority rule on heterogeneous networks

    International Nuclear Information System (INIS)

    Lambiotte, R

    2008-01-01

    We focus on the majority rule (MR) applied on heterogeneous networks. When the underlying topology is homogeneous, the system is shown to exhibit a transition from an ordered regime to a disordered regime when the noise is increased. When the network exhibits modular structures, in contrast, the system may also exhibit an asymmetric regime, where the nodes in each community reach an opposite average opinion. Finally, the node degree heterogeneity is shown to play an important role by displacing the location of the order-disorder transition and by making the system exhibit non-equipartition of the average spin

  8. Societal risk and major disasters

    International Nuclear Information System (INIS)

    Clement, C.F.

    1989-01-01

    A disaster can be defined as an event, or a series of events, in which a large number of people is adversely affected by a single cause. This definition includes man-made accidents, like that at Chernobyl, as well as the natural disasters that insurance companies are sometimes pleased to describe as Acts of God. In 1986 alone, 12,000 people died and 2.2 million were made homeless by 215 major accidents or disasters. The nature of risk is examined in this paper. (author)

  9. Detection of protein complex from protein-protein interaction network using Markov clustering

    International Nuclear Information System (INIS)

    Ochieng, P J; Kusuma, W A; Haryanto, T

    2017-01-01

    Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks. (paper)

  10. Dietary protein and blood pressure : epidemiological studies

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.

    2012-01-01


    Background
    Elevated blood pressure is a major risk factor for cardiovascular diseases. Diet and lifestyle have a substantial impact on blood pressure, but the role of protein intake is not yet clear. This thesis focuses on total dietary protein, types of protein (i.e. plant and

  11. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  12. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  13. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  14. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

  15. DNA double-strand break repair: a tale of pathway choices

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Xingzhi Xu

    2016-01-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways.DSB repair is critical for genome integrity,cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy.The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts.Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages,downstream effects,and distinct chromosomal histone marks.These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

  16. Major Environmental Policy in 2000

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hong Jin [Ministry Of Environment, Kwachon (Korea)

    2000-04-01

    As a new millennium has started, there are active movements developing a basic paradigm of vision and policy over a nation-wide to prepare changes actively. For the environmental sector, it is possible to live in a pleasant environment if everyone prepare and work together like dealing with Y2K problem. With a goal of being an environmentally advanced country in the early new millennium, it is planned to improve a basic life environment such as water and air and to promote an advanced environmental management policy for showing results of its reform in 2000. Therefore, it examines environmental management circumstances and a direction of environmental policy first and it discusses more about major environmental policy related to petroleum industry. 7 tabs.

  17. Neuroticism in remitted major depression

    DEFF Research Database (Denmark)

    Gade, Anders; Kristoffersen, Marius; Kessing, Lars Vedel

    2015-01-01

    not been consistent. METHOD: We examined neuroticism, extraversion and perceived stress in 88 fully remitted depressed patients with a mean age of 60 years and with a history of hospitalization for major depressive disorder. Patients were divided into those with onset after and those with onset before 50......BACKGROUND: The personality trait of neuroticism is strongly related to depression, but depression is etiologically heterogeneous. Late-onset depression (LOD) may be more closely related to vascular factors, and previous studies of neuroticism in LOD versus early-onset depression (EOD) have...... age of onset and neuroticism was confirmed in analyses based on age of depression onset as a continuous variable. CONCLUSION: Neuroticism may be an etiological factor in EOD but not or less so in LOD. This finding contributes to the growing evidence for etiological differences between early- and late...

  18. Psychosocial implications of Thalassemia Major.

    Science.gov (United States)

    Aydinok, Yesim; Erermis, Serpil; Bukusoglu, Nagihan; Yilmaz, Deniz; Solak, Ufuk

    2005-02-01

    Many causes including the chronicity of disease, burden of treatment modalities, morbidities, and the expectation of early death resulting from the disease complications, may lead to psychosocial burden in Thalassemia Major (TM) patients. A total of 38 patients with TM and their mothers were recruited to evaluate the psychosocial burden as well as to disclose whether the psychological status of the patients contribute to the compliance with the therapy or to the contrary. Demographic and disease variables were obtained. Child Behavior Check-list (CBCL) was completed by the mothers of the patients. A detailed psychiatric interview based on the 4th edition of the Diagnostic and Statistical Manual diagnostic criteria was performed for each patient. Symptom Distress Checklist 90 (SCL-90) scale was given to all mothers for evaluating their psychopathology. Although CBCL scores remained between the normal ranges, desferrioxamine mesylate (DFO)-compliant patients and the patients with lower ferritin values had significantly higher scores. A total of 24% of the patients had a psychiatric diagnosis including major depression, anxiety disorder, tic disorder, and enuresis nocturnal. The psychiatric diagnosis was significantly higher in the patients who were compliant with desferrioxamine compared with the non-compliant group (P = 0.007). The SCL-90 scores indicated that the mothers who had a child with good adherence to DFO had higher scale scores than the mothers with a poor adherent child. The increase risk of psychosocial and behavioral problems in thalassemics and their parents indicated the importance of a lifelong psychosocial support for the prevention of mental health issues. The patients and their parents, who were more conscious of the illness, were more worried but more compliant with the therapy and need stronger psychiatric support.

  19. Thalassaemia major and the heart

    Directory of Open Access Journals (Sweden)

    J. Malcolm Walker

    2013-03-01

    Full Text Available Disorders of haemoglobin synthesis are the commonest monogenetic disorders worldwide. When first described, thalassaemia was universally fatal in childhood, but after the adoption of regular blood transfusion survival until early teenage and adulthood was to be expected. At that stage in the life of these affected individuals organ failure followed, due to accumulated iron, for which the human has no excretory capacity. Principal amongst the tissues affected by iron overload is the heart and even to the present day, heart disease accounts for the overwhelming majority of premature deaths in this population. Managing transfusion derived iron overload was the next hurdle for clinicians and the families of the patients. For nearly four decades the only available treatment was the demanding regime of parenteral chelation therapy, required on a daily basis, to achieve growth, development and survival with limited or no organ damage. Despite the adoption of these treatment strategies the outlook for thalassaemia patients remained poor, with a 30% to 40% mortality occurring between late teenage and 30 years of age, even in well organised health care systems, such as in the UK, where regular transfusion and desferioxamine treatment were readily available. This dreadful early mortality, largely as a consequence of myocardial iron overload, (1,2 is now improving so that in the UK and other developed nations, heart failure in thalassaemic patients has become uncommon and premature death a much rarer tragedy. This editorial reviews, from a personal viewpoint of a cardiologist involved in the care of these patients for the last 20 years, the progress in the management of the cardiovascular complications of thalassaemia major (TM, which has followed better techniques of identifying those thalassaemic individuals at greatest risk, improved chelation strategies making best use of the three chelating agents that are now available and improved co

  20. Hepatitis C virus infection protein network.

    Science.gov (United States)

    de Chassey, B; Navratil, V; Tafforeau, L; Hiet, M S; Aublin-Gex, A; Agaugué, S; Meiffren, G; Pradezynski, F; Faria, B F; Chantier, T; Le Breton, M; Pellet, J; Davoust, N; Mangeot, P E; Chaboud, A; Penin, F; Jacob, Y; Vidalain, P O; Vidal, M; André, P; Rabourdin-Combe, C; Lotteau, V

    2008-01-01

    A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

  1. Tilecal meets two major milestones

    CERN Multimedia

    Cavalli-Sforza, M.

    Over the last two months the Tile Calorimeter passed not one but two major milestones. In early May, the last of the 64 modules that make up one of the two Extended Barrels arrived at CERN from IFAE-Barcelona, equipped with optical components and tested. And during the Overview Week in Clermont-Ferrand, the last of the 64 Barrel modules, mechanically assembled, arrived from JINR-Dubna. Just a brief reminder: the ATLAS Tile Calorimeter is composed of 3 cylinders ("barrels") of steel, scintillating tiles and optical fibers, altogether about 12 m long, with an outer diameter of 8.4 m, and weighing about 2700 tons. The central cavity will contain the Liquid Argon cryostats, and the whole calorimetry system will measure the direction and energy of jets produced at the LHC, as well as the missing transverse energy, which as everyone knows is one of the telltale signals of new and exciting physics. Each of the three cylinders is divided azimuthally into 64 modules - much like the slices of an orange. The modules ar...

  2. Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

    Directory of Open Access Journals (Sweden)

    Ruzianisra Mohamed

    Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

  3. Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome

    Science.gov (United States)

    Kosová, Klára; Vítámvás, Pavel; Urban, Milan O.; Prášil, Ilja T.; Renaut, Jenny

    2018-01-01

    HIGHLIGHTS: Major environmental and genetic factors determining stress-related protein abundance are discussed.Major aspects of protein biological function including protein isoforms and PTMs, cellular localization and protein interactions are discussed.Functional diversity of protein isoforms and PTMs is discussed. Abiotic stresses reveal profound impacts on plant proteomes including alterations in protein relative abundance, cellular localization, post-transcriptional and post-translational modifications (PTMs), protein interactions with other protein partners, and, finally, protein biological functions. The main aim of the present review is to discuss the major factors determining stress-related protein accumulation and their final biological functions. A dynamics of stress response including stress acclimation to altered ambient conditions and recovery after the stress treatment is discussed. The results of proteomic studies aimed at a comparison of stress response in plant genotypes differing in stress adaptability reveal constitutively enhanced levels of several stress-related proteins (protective proteins, chaperones, ROS scavenging- and detoxification-related enzymes) in the tolerant genotypes with respect to the susceptible ones. Tolerant genotypes can efficiently adjust energy metabolism to enhanced needs during stress acclimation. Stress tolerance vs. stress susceptibility are relative terms which can reflect different stress-coping strategies depending on the given stress treatment. The role of differential protein isoforms and PTMs with respect to their biological functions in different physiological constraints (cellular compartments and interacting partners) is discussed. The importance of protein functional studies following high-throughput proteome analyses is presented in a broader context of plant biology. In summary, the manuscript tries to provide an overview of the major factors which have to be considered when interpreting data from proteomic

  4. Major new sources of biological ice nuclei

    Science.gov (United States)

    Moffett, B. F.; Hill, T.; Henderson-Begg, S. K.

    2009-12-01

    Almost all research on biological ice nucleation has focussed on a limited number of bacteria. Here we characterise several major new sources of biogenic ice nuclei. These include mosses, hornworts, liverworts and cyanobacteria. Ice nucleation in the eukaryotic bryophytes appears to be ubiquitous. The temperature at which these organisms nucleate is that at which the difference in vapour pressure over ice and water is at or close to its maximum. At these temperatures (-8 to -18 degrees C) ice will grow at the expense of supercooled water. These organisms are dependent for their water on occult precipitation - fog, dew and cloudwater which by its nature is not collected in conventional rain gauges. Therefore we suggest that these organism produce ice nuclei as a water harvesting mechanism. Since the same mechanism would also drive the Bergeron-Findeisen process, and as moss is known to become airborne, these nuclei may have a role in the initiation of precipitation. The properties of these ice nuclei are very different from the well characterised bacterial nuclei. We will also present DNA sequence data showing that, although related, the proteins responsible are only very distantly related to the classical bacterial ice nuclei.

  5. The inflammatory cytokines: molecular biomarkers for major depressive disorder?

    Science.gov (United States)

    Martin, Charlotte; Tansey, Katherine E; Schalkwyk, Leonard C; Powell, Timothy R

    2015-01-01

    Cytokines are pleotropic cell signaling proteins that, in addition to their role as inflammatory mediators, also affect neurotransmitter systems, brain functionality and mood. Here we explore the potential utility of cytokine biomarkers for major depressive disorder. Specifically, we explore how genetic, transcriptomic and proteomic information relating to the cytokines might act as biomarkers, aiding clinical diagnosis and treatment selection processes. We advise future studies to investigate whether cytokine biomarkers might differentiate major depressive disorder patients from other patient groups with overlapping clinical characteristics. Furthermore, we invite future pharmacogenetic studies to investigate whether early antidepressant-induced changes to cytokine mRNA or protein levels precede behavioral changes and act as longer-term predictors of clinical antidepressant response.

  6. Intercellular protein-protein interactions at synapses.

    Science.gov (United States)

    Yang, Xiaofei; Hou, Dongmei; Jiang, Wei; Zhang, Chen

    2014-06-01

    Chemical synapses are asymmetric intercellular junctions through which neurons send nerve impulses to communicate with other neurons or excitable cells. The appropriate formation of synapses, both spatially and temporally, is essential for brain function and depends on the intercellular protein-protein interactions of cell adhesion molecules (CAMs) at synaptic clefts. The CAM proteins link pre- and post-synaptic sites, and play essential roles in promoting synapse formation and maturation, maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. Alteration of the interactions of CAMs leads to structural and functional impairments, which results in many neurological disorders, such as autism, Alzheimer's disease and schizophrenia. Therefore, it is crucial to understand the functions of CAMs during development and in the mature neural system, as well as in the pathogenesis of some neurological disorders. Here, we review the function of the major classes of CAMs, and how dysfunction of CAMs relates to several neurological disorders.

  7. Protein function prediction using neighbor relativity in protein-protein interaction network.

    Science.gov (United States)

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. [Oromaxillofacial changes in thalassemia major].

    Science.gov (United States)

    De Mattia, D; Pettini, P L; Sabato, V; Rubini, G; Laforgia, A; Schettini, F

    1996-01-01

    Sixty patients (31 male and 29 female) with thalassemia major, aged between 6 and 26 years, 18 of which were splenectomized, were observed in this study evaluating the oro-maxillo-facial alterations and correlating them to transfusion indexes, serum ferritin levels, splenectomy and age. For each patient a haematologic and odontostomatologic card was filed with a view to report the medical and clinical history regarding: the haematologic picture, the prevention of caries and parodontal disease, the facies characteristics, the odonto-stomatologic examination, the orthodontic diagnosis, the skull X-rays and the orthopantomography. Poor oral hygiene as well as misknowledge of prevention were generally observed. All the patients showed carious lesions but most of them had never seen a dentist for therapy. The disharmonious growth of splanchnocranium, with the enlargement of the jaw and of its alveolar process, induced by the bone marrow hyperplasia, produced various and serious malocclusion stages (Angle's II class, deep bite, open bite), gnathologic alterations, hypodiaphanous paranasal sinuses and orbital hypertelorism, with a typical oriental-like facies. Malocclusion and the poor oral hygienic conditions determined the occurrence of marginal gingivitis, mainly localized at the level of the lower frontal teeth. In only 3 patients the oral mucous membrane was pale and atrophic. During this investigation agenesia and dental retention were reported in 30% and in 26% of the examined cases respectively, while no patients had supernumerary teeth. Tooth volume, position and shape abnormalities rarely occurred. Only in two patients was enamel hypoplasia described. The caries frequency greatly varied in number and in degree. Only five patients did not show any carious lesions. The caries index (DMF) for the permanent teeth calculated in all the 60 subjects was 5, 12 +/- 4.76. By utilizing Spearman's rank test the number of teeth with caries in the permanent dentition (DFM

  9. Post-Electrophoretic Identification of Oxidized Proteins

    Science.gov (United States)

    Conrad, Craig C; Talent, John M; Malakowsky, Christina A

    1999-01-01

    The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold. PMID:12734585

  10. Major advances in applied dairy cattle nutrition.

    Science.gov (United States)

    Eastridge, M L

    2006-04-01

    Milk yield per cow continues to increase with a slower rate of increase in dry matter intake; thus, efficiency of ruminal fermentation and digestibility of the dietary components are key factors in improving the efficiency of feed use. Over the past 25 yr, at least 2,567 articles relating to ruminant or dairy nutrition have been published in the Journal of Dairy Science. These studies have provided important advancements in improving feed efficiency and animal health by improving quality of feeds, increasing feedstuff and overall diet digestibility, better defining interactions among feedstuffs in diets, identifying alternative feed ingredients, better defining nutrient requirements, and improving efficiency of ruminal fermentation. The publications are vital in continuing to make advancements in providing adequate nutrition to dairy cattle and for facilitating exchange of knowledge among scientists. Forages have been studied more extensively than any other type of feed. Cereal grains continue to be the primary contributors of starch to diets, and thus are very important in meeting the energy needs of dairy cattle. Processing of cereal grains has improved their use. Feeding by-products contributes valuable nutrients to diets and allows feedstuffs to be used that would otherwise be handled as wastes in landfills. Many of these by-products provide a considerable amount of protein, nonforage fiber, fat, and minerals (sometimes a detriment as in the case of P) to diets. The primary feeding system today is the total mixed ration, with still considerable use of the pasture system. Major improvements have occurred in the use of protein, carbohydrates, and fats in diets. Although advancements have been made in feeding practices to minimize the risk of metabolic diseases, the periparturient period continues to present some of the greatest challenges in animal health. Computers are a must today for diet formulation and evaluation, but fewer software programs are developed by

  11. Computational protein design: a review

    International Nuclear Information System (INIS)

    Coluzza, Ivan

    2017-01-01

    Proteins are one of the most versatile modular assembling systems in nature. Experimentally, more than 110 000 protein structures have been identified and more are deposited every day in the Protein Data Bank. Such an enormous structural variety is to a first approximation controlled by the sequence of amino acids along the peptide chain of each protein. Understanding how the structural and functional properties of the target can be encoded in this sequence is the main objective of protein design. Unfortunately, rational protein design remains one of the major challenges across the disciplines of biology, physics and chemistry. The implications of solving this problem are enormous and branch into materials science, drug design, evolution and even cryptography. For instance, in the field of drug design an effective computational method to design protein-based ligands for biological targets such as viruses, bacteria or tumour cells, could give a significant boost to the development of new therapies with reduced side effects. In materials science, self-assembly is a highly desired property and soon artificial proteins could represent a new class of designable self-assembling materials. The scope of this review is to describe the state of the art in computational protein design methods and give the reader an outline of what developments could be expected in the near future. (topical review)

  12. Progress on major genes for high fecundity in ewes

    Directory of Open Access Journals (Sweden)

    Qiuyue LIU,Zhangyuan PAN,Xiangyu WANG,Wenping HU,Ran DI,Yaxing YAO,Mingxing CHU

    2014-12-01

    Full Text Available The existence of major genes affecting fecundity in sheep flocks throughout the world has been demonstrated. Three major genes whose mutations can increase ovulation rate have been discovered, and all related to the transforming growth factor β (TGF-β superfamily. The mutant FecB of bone morphogenetic protein receptor 1B (BMPR1B has an additive effect on ovulation rate. Six mutations (FecXI, FecXH, FecXG, FecXB, FecXL, FecXR of bone morphogenetic protein 15 (BMP15 related with fertility have been identified that share the same mechanism. All the mutants can increase ovulation rate in heterozygotes and cause complete sterility in homozygotes. Homozygous ewes with two new mutations (FecXGr, FecXO of BMP15 had increased ovulation rate without causing sterility. There are five mutations in growth differentiation factor 9 (GDF9 associated with sheep prolificacy where FecGE and FecGF have additive an effect on ovulation rate and litter size. The newly identified β-1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2 gene of FecL is proposed as a new mechanism of ovulation rate regulation in sheep. Woodlands is an X-linked maternally imprinted gene which increases ovulation rate. In addition, several putative major genes need to be verified. This review is focused on the identification of the mutations and mechanisms whereby the major genes affecting ovulation rate.

  13. Hot-spot analysis for drug discovery targeting protein-protein interactions.

    Science.gov (United States)

    Rosell, Mireia; Fernández-Recio, Juan

    2018-04-01

    Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

  14. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    International Nuclear Information System (INIS)

    Dooley, J.S.G.; Trust, T.J.

    1988-01-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125 I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to 125 I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein

  15. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C

    1993-01-01

    hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development......The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus...... blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved...

  16. A major genetic component of BSE susceptibility

    Science.gov (United States)

    Juling, Katrin; Schwarzenbacher, Hermann; Williams, John L; Fries, Ruedi

    2006-01-01

    Background Coding variants of the prion protein gene (PRNP) have been shown to be major determinants for the susceptibility to transmitted prion diseases in humans, mice and sheep. However, to date, the effects of polymorphisms in the coding and regulatory regions of bovine PRNP on bovine spongiform encephalopathy (BSE) susceptibility have been considered marginal or non-existent. Here we analysed two insertion/deletion (indel) polymorphisms in the regulatory region of bovine PRNP in BSE affected animals and controls of four independent cattle populations from UK and Germany. Results In the present report, we show that two previously reported 23- and 12-bp insertion/deletion (indel) polymorphisms in the regulatory region of bovine PRNP are strongly associated with BSE incidence in cattle. Genotyping of BSE-affected and control animals of UK Holstein, German Holstein, German Brown and German Fleckvieh breeds revealed a significant overrepresentation of the deletion alleles at both polymorphic sites in diseased animals (P = 2.01 × 10-3 and P = 8.66 × 10-5, respectively). The main effect on susceptibility is associated with the 12-bp indel polymorphism. Compared with non-carriers, heterozygous and homozygous carriers of the 12-bp deletion allele possess relatively higher risks of having BSE, ranging from 1.32 to 4.01 and 1.74 to 3.65 in the different breeds. These values correspond to population attributable risks ranging from 35% to 53%. Conclusion Our results demonstrate a substantial genetic PRNP associated component for BSE susceptibility in cattle. Although the BSE risk conferred by the deletion allele of the 12-bp indel in the regulatory region of PRNP is substantial, the main risk factor for BSE in cattle is environmental, i.e. exposure to feedstuffs contaminated with the infectious agent. PMID:17014722

  17. A major genetic component of BSE susceptibility

    Directory of Open Access Journals (Sweden)

    Williams John L

    2006-10-01

    Full Text Available Abstract Background Coding variants of the prion protein gene (PRNP have been shown to be major determinants for the susceptibility to transmitted prion diseases in humans, mice and sheep. However, to date, the effects of polymorphisms in the coding and regulatory regions of bovine PRNP on bovine spongiform encephalopathy (BSE susceptibility have been considered marginal or non-existent. Here we analysed two insertion/deletion (indel polymorphisms in the regulatory region of bovine PRNP in BSE affected animals and controls of four independent cattle populations from UK and Germany. Results In the present report, we show that two previously reported 23- and 12-bp insertion/deletion (indel polymorphisms in the regulatory region of bovine PRNP are strongly associated with BSE incidence in cattle. Genotyping of BSE-affected and control animals of UK Holstein, German Holstein, German Brown and German Fleckvieh breeds revealed a significant overrepresentation of the deletion alleles at both polymorphic sites in diseased animals (P = 2.01 × 10-3 and P = 8.66 × 10-5, respectively. The main effect on susceptibility is associated with the 12-bp indel polymorphism. Compared with non-carriers, heterozygous and homozygous carriers of the 12-bp deletion allele possess relatively higher risks of having BSE, ranging from 1.32 to 4.01 and 1.74 to 3.65 in the different breeds. These values correspond to population attributable risks ranging from 35% to 53%. Conclusion Our results demonstrate a substantial genetic PRNP associated component for BSE susceptibility in cattle. Although the BSE risk conferred by the deletion allele of the 12-bp indel in the regulatory region of PRNP is substantial, the main risk factor for BSE in cattle is environmental, i.e. exposure to feedstuffs contaminated with the infectious agent.

  18. Major advances in fundamental dairy cattle nutrition.

    Science.gov (United States)

    Drackley, J K; Donkin, S S; Reynolds, C K

    2006-04-01

    Fundamental nutrition seeks to describe the complex biochemical reactions involved in assimilation and processing of nutrients by various tissues and organs, and to quantify nutrient movement (flux) through those processes. Over the last 25 yr, considerable progress has been made in increasing our understanding of metabolism in dairy cattle. Major advances have been made at all levels of biological organization, including the whole animal, organ systems, tissues, cells, and molecules. At the whole-animal level, progress has been made in delineating metabolism during late pregnancy and the transition to lactation, as well as in whole-body use of energy-yielding substrates and amino acids for growth in young calves. An explosion of research using multicatheterization techniques has led to better quantitative descriptions of nutrient use by tissues of the portal-drained viscera (digestive tract, pancreas, and associated adipose tissues) and liver. Isolated tissue preparations have provided important information on the interrelationships among glucose, fatty acid, and amino acid metabolism in liver, adipose tissue, and mammary gland, as well as the regulation of these pathways during different physiological states. Finally, the last 25 yr has witnessed the birth of "molecular biology" approaches to understanding fundamental nutrition. Although measurements of mRNA abundance for proteins of interest already have provided new insights into regulation of metabolism, the next 25 yr will likely see remarkable advances as these techniques continue to be applied to problems of dairy cattle biology. Integration of the "omics" technologies (functional genomics, proteomics, and metabolomics) with measurements of tissue metabolism obtained by other methods is a particularly exciting prospect for the future. The result should be improved animal health and well being, more efficient dairy production, and better models to predict nutritional requirements and provide rations to meet

  19. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways.

    Science.gov (United States)

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S

    2011-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. © 2010 Blackwell Publishing Ltd.

  20. Fish allergens at a glance: Variable allergenicity of parvalbumins, the major fish allergens

    OpenAIRE

    Annette eKuehn; Ines eSwoboda; Karthik eArumugam; Christiane eHilger; François eHentges; François eHentges

    2014-01-01

    Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand,...

  1. Fish Allergens at a Glance: Variable Allergenicity of Parvalbumins, the Major Fish Allergens

    OpenAIRE

    Kuehn, Annette; Swoboda, Ines; Arumugam, Karthik; Hilger, Christiane; Hentges, François

    2014-01-01

    Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand,...

  2. Tuber storage proteins.

    Science.gov (United States)

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  3. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  4. BCR/ABL downregulates DNA-PK(CS)-dependent and upregulates backup non-homologous end joining in leukemic cells.

    Science.gov (United States)

    Poplawski, Tomasz; Blasiak, Janusz

    2010-06-01

    Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK(CS); D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK(CS) (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK(CS), did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.

  5. Identification of parvalbumin and two new thermolabile major allergens of Thunnus tonggol using a proteomics approach.

    Science.gov (United States)

    Rosmilah, Misnan; Shahnaz, Murad; Meinir, Jones; Masita, Arip; Noormalin, Abdullah; Jamaluddin, Mohamed

    2013-01-01

    The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach. Raw and boiled extracts of the fish were prepared. Fish proteins were separated by means of SDS-PAGE and two-dimensional (2-DE) electrophoresis. 1-DE immunoblotting of raw extract was performed with sera from fish-allergic patients. Ten sera were further analysed by 2-DE immunoblotting. Selected major allergenic protein spots were excised, trypsin digested and analysed by means of mass spectrometry. SDS-PAGE of raw extract revealed 26 protein fractions, while boiled extract demonstrated fewer bands. The 2-DE gel profile of the raw extract further fractionated the protein bands to more than 100 distinct protein spots. 1-DE immunoblotting of raw extract exhibited two thermolabile protein fractions of 42 and 51 kDa as the major allergens, while the boiled extract only revealed a single IgE-binding band at 151 kDa. 2-DE immunoblotting of raw extract further detected numerous major IgE-reactive spots of 11-13, 42 and 51 kDa. Mass spectrometry analysis of the peptides generated from the 12, 42 and 51 kDa digested spots indicated that these spots were parvalbumin, creatine kinase and enolase, respectively. In addition to parvalbumin, two new thermolabile allergens were identified as major allergenic proteins of T. tonggol. This study proved that both thermostable and thermolabile proteins are important in local tuna allergy and should be included in diagnostic strategies.

  6. Protein supplementation with sports protein bars in renal patients.

    Science.gov (United States)

    Meade, Anthony

    2007-05-01

    bars as the preferred supplement. The major reasons for not continuing were taste and chewability, especially in older patients with dentures. Serum potassium and phosphate levels were not increased with supplementation. Measured serum albumin and protein catabolic rate were considered unreliable indicators because not all patients were medically stable. Sports protein bars are an acceptable protein and energy supplement for patients on hemodialysis. Sports protein bars are well accepted by patients except when dentures limit chewability. Sports protein bars have advantages over fluid-based supplements in patients with fluid restrictions.

  7. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  8. Detecting protein complexes based on a combination of topological and biological properties in protein-protein interaction network

    Directory of Open Access Journals (Sweden)

    Pooja Sharma

    2018-06-01

    Full Text Available Protein complexes are known to play a major role in controlling cellular activity in a living being. Identifying complexes from raw protein protein interactions (PPIs is an important area of research. Earlier work has been limited mostly to yeast. Such protein complex identification methods, when applied to large human PPIs often give poor performance. We introduce a novel method called CSC to detect protein complexes. The method is evaluated in terms of positive predictive value, sensitivity and accuracy using the datasets of the model organism, yeast and humans. CSC outperforms several other competing algorithms for both organisms. Further, we present a framework to establish the usefulness of CSC in analyzing the influence of a given disease gene in a complex topologically as well as biologically considering eight major association factors. Keywords: Protein complex, Connectivity, Semantic similarity, Contribution

  9. Major Highway Lines, US, 2015, NAVTEQ

    Data.gov (United States)

    U.S. Environmental Protection Agency — NAVTEQ Major Highways for the United States. The Major Highways layer contains Road Network features based on the Functional Class attribute value on each link...

  10. Functional assignment to JEV proteins using SVM.

    Science.gov (United States)

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

  11. A tripartite approach identifies the major sunflower seed albumins.

    Science.gov (United States)

    Jayasena, Achala S; Franke, Bastian; Rosengren, Johan; Mylne, Joshua S

    2016-03-01

    We have used a combination of genomic, transcriptomic, and proteomic approaches to identify the napin-type albumin genes in sunflower and define their contributions to the seed albumin pool. Seed protein content is determined by the expression of what are typically large gene families. A major class of seed storage proteins is the napin-type, water soluble albumins. In this work we provide a comprehensive analysis of the napin-type albumin content of the common sunflower (Helianthus annuus) by analyzing a draft genome, a transcriptome and performing a proteomic analysis of the seed albumin fraction. We show that although sunflower contains at least 26 genes for napin-type albumins, only 15 of these are present at the mRNA level. We found protein evidence for 11 of these but the albumin content of mature seeds is dominated by the encoded products of just three genes. So despite high genetic redundancy for albumins, only a small sub-set of this gene family contributes to total seed albumin content. The three genes identified as producing the majority of sunflower seed albumin are potential future candidates for manipulation through genetics and breeding.

  12. Molecular evolution of the major chemosensory gene families in insects.

    Science.gov (United States)

    Sánchez-Gracia, A; Vieira, F G; Rozas, J

    2009-09-01

    Chemoreception is a crucial biological process that is essential for the survival of animals. In insects, olfaction allows the organism to recognise volatile cues that allow the detection of food, predators and mates, whereas the sense of taste commonly allows the discrimination of soluble stimulants that elicit feeding behaviours and can also initiate innate sexual and reproductive responses. The most important proteins involved in the recognition of chemical cues comprise moderately sized multigene families. These families include odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), which are involved in peripheral olfactory processing, and the chemoreceptor superfamily formed by the olfactory receptor (OR) and gustatory receptor (GR) families. Here, we review some recent evolutionary genomic studies of chemosensory gene families using the data from fully sequenced insect genomes, especially from the 12 newly available Drosophila genomes. Overall, the results clearly support the birth-and-death model as the major mechanism of evolution in these gene families. Namely, new members arise by tandem gene duplication, progressively diverge in sequence and function, and can eventually be lost from the genome by a deletion or pseudogenisation event. Adaptive changes fostered by environmental shifts are also observed in the evolution of chemosensory families in insects and likely involve reproductive, ecological or behavioural traits. Consequently, the current size of these gene families is mainly a result of random gene gain and loss events. This dynamic process may represent a major source of genetic variation, providing opportunities for FUTURE specific adaptations.

  13. Text Mining for Protein Docking.

    Directory of Open Access Journals (Sweden)

    Varsha D Badal

    2015-12-01

    Full Text Available The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking. Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu. The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound

  14. Why It Pays to Major in Economics

    Science.gov (United States)

    Carroll, Thomas; Assane, Djeto; Busker, Jared

    2014-01-01

    In this article, the authors use a large, recent, and accessible data set to examine the effect of economics major on individual earnings. They find a significant positive earnings gain for economics majors relative to other majors, and this advantage increases with the level of education. Their findings are consistent with Black, Sanders, and…

  15. 75 FR 31383 - Major Capital Investment Projects

    Science.gov (United States)

    2010-06-03

    ...-0009] RIN 2132-AB02 Major Capital Investment Projects AGENCIES: Federal Transit Administration (FTA... current approach to evaluating and rating major capital investment projects (``New Starts'' and ``Small...'' to address identified transportation needs in the corridor without a major capital investment in new...

  16. Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

    Science.gov (United States)

    Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih-Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung-Jong; Chen, David J; Harrison, Lynn

    2011-11-01

    Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

  17. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    Science.gov (United States)

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Casein - whey protein interactions in heated milk

    NARCIS (Netherlands)

    Vasbinder, Astrid Jolanda

    2002-01-01

    Heating of milk is an essential step in the processing of various dairy products, like for example yoghurt. A major consequence of the heat treatment is the denaturation of whey proteins, which either associate with the casein micelle or form soluble whey protein aggregates. By combination of

  19. Getting out : protein traffic in prokaryotes

    NARCIS (Netherlands)

    Pugsley, A.P; Francetic, O; Driessen, A.J.M.; de Lorenzo, V.

    Protein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for 'more complex' processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert

  20. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  1. The Prototypical Majority Effect Under Social Influence.

    Science.gov (United States)

    Koriat, Asher; Adiv-Mashinsky, Shiri; Undorf, Monika; Schwarz, Norbert

    2018-05-01

    Majority views are reported with greater confidence and fluency than minority views, with the difference increasing with majority size. This Prototypical Majority Effect (PME) was attributed generally to conformity pressure, but Koriat et al. showed that it can arise from the processes underlying decision and confidence independent of social influence. Here we examined the PME under conditions that differ in social influence. In Experiment 1, a robust PME emerged in the absence of information about the majority views, but the provision sof that information increased the choice of the majority view and magnified the PME. In Experiment 2, a PME emerged in a minority-biased condition that misled participants to believe that the majority view was the minority view, but the PME was stronger in a majority-biased condition. The results were discussed in terms of a dual-process view: The PME observed under social influence may contain externally driven and internally driven components.

  2. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  3. Statistical Analysis of Protein Ensembles

    Science.gov (United States)

    Máté, Gabriell; Heermann, Dieter

    2014-04-01

    As 3D protein-configuration data is piling up, there is an ever-increasing need for well-defined, mathematically rigorous analysis approaches, especially that the vast majority of the currently available methods rely heavily on heuristics. We propose an analysis framework which stems from topology, the field of mathematics which studies properties preserved under continuous deformations. First, we calculate a barcode representation of the molecules employing computational topology algorithms. Bars in this barcode represent different topological features. Molecules are compared through their barcodes by statistically determining the difference in the set of their topological features. As a proof-of-principle application, we analyze a dataset compiled of ensembles of different proteins, obtained from the Ensemble Protein Database. We demonstrate that our approach correctly detects the different protein groupings.

  4. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

  5. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

    NARCIS (Netherlands)

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

    2016-01-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1

  6. DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity

    DEFF Research Database (Denmark)

    Gupta, Rajat; Somyajit, Kumar; Narita, Takeo

    2018-01-01

    Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expres...... and the evolution of antibody CSR in higher vertebrates....

  7. Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA.

    Science.gov (United States)

    Zelensky, Alex N; Schimmel, Joost; Kool, Hanneke; Kanaar, Roland; Tijsterman, Marcel

    2017-07-07

    Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

  8. Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins

    International Nuclear Information System (INIS)

    Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

    1987-01-01

    Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by 125 I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml

  9. Secretory proteins of the pulmonary extracellular lining

    International Nuclear Information System (INIS)

    Gupta, R.P.; Patton, S.E.; Eddy, M.; Smits, H.L.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.R.

    1986-01-01

    The objective of this investigation was to identify proteins in the pulmonary extracellular lining (EL) that are secreted by cells of the pulmonary epithelium. Pulmonary lavage effluents from the lungs of rabbits were centrifuged to remove all cells and particulate materials. Serum proteins were removed by repeatedly passing concentrated lavage effluent fluid through an affinity column containing IgG fraction of goat anti-rabbit (whole serum) antiserum bound to Sepharose-4B. Nonserum proteins accounted for 21.3 +/- 10.3% of the total soluble proteins in pulmonary lavage effluents. Serum free lavage effluents (SFL) contained 25 identifiable proteins as determined by using SDS-PAGE under reducing conditions. Of these proteins approximately 73% was accounted for by a single protein with MW of 66 kd. The secretory nature of the proteins present in SFL was investigated by studying the incorporation of 35 S-methionine into proteins released by lung slices and trachea followed by SDS-PAGE and autoradiography. Many, but not all proteins present in SFL were identified as proteins secreted by pulmonary tissues. The major secretory proteins appeared to have MWs of 59, 53, 48, 43, 24, 14, and 6 kd under reducing conditions. These data demonstrate the presence of several proteins in the pulmonary extracellular lining that appear to be secreted by the pulmonary epithelium

  10. Protein improvement in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Rabson, R

    1974-07-01

    There are compelling reasons for attempting to increase the quality and quantity of protein available in crop plants through plant breeding, despite the fact that some critics have argued that no worldwide protein shortage exists. What used to be thought of as a 'protein gap' has now come to be considered in terms of protein-calorie malnutrition. This is only right since protein and calorie nutrition are inextricable. t the moment there are still unanswered questions as to the precise protein requirements of humans as a function of age, health and ambient conditions. There are, in addition, some indications that the incidence of Kwashiorkor (protein deficiency disease) is increasing in different parts of the world. At a recent meeting of the Protein Advisory Group of the United Nations System, Dr. Jean Mayer, an eminent human nutritionist of Harvard University, U.S.A., indicated the reasons for concern for the current food situation generally, and the protein food supply in particular. These factors include: - Immoderate continuing human population increases, most pronounced in some poor developing countries. - The highly accelerated consumption of animal foods associated with increasing affluence in the richer countries of the world. The production of such foods as meat demands great expenditures of grain, which is an inefficient mode of obtaining the required calories and protein for human consumption. - The over-exploitation of many of the world's fishery resources resulting in reduced yields, perhaps irreversibly, of some fishes. - Recent price increases in petroleum and fertilizer products which have imposed a major obstacle to increasing crop production. - The apparent alteration of climates in places like Africa, Asia and other parts of the Northern hemisphere which may put significant restrictions on crop production. hey are cogent reasons to be seriously concerned about these matters. (author)

  11. Protein improvement in crop plants

    International Nuclear Information System (INIS)

    Rabson, R.

    1974-01-01

    There are compelling reasons for attempting to increase the quality and quantity of protein available in crop plants through plant breeding, despite the fact that some critics have argued that no worldwide protein shortage exists. What used to be thought of as a 'protein gap' has now come to be considered in terms of protein-calorie malnutrition. This is only right since protein and calorie nutrition are inextricable. t the moment there are still unanswered questions as to the precise protein requirements of humans as a function of age, health and ambient conditions. There are, in addition, some indications that the incidence of Kwashiorkor (protein deficiency disease) is increasing in different parts of the world. At a recent meeting of the Protein Advisory Group of the United Nations System, Dr. Jean Mayer, an eminent human nutritionist of Harvard University, U.S.A., indicated the reasons for concern for the current food situation generally, and the protein food supply in particular. These factors include: - Immoderate continuing human population increases, most pronounced in some poor developing countries. - The highly accelerated consumption of animal foods associated with increasing affluence in the richer countries of the world. The production of such foods as meat demands great expenditures of grain, which is an inefficient mode of obtaining the required calories and protein for human consumption. - The over-exploitation of many of the world's fishery resources resulting in reduced yields, perhaps irreversibly, of some fishes. - Recent price increases in petroleum and fertilizer products which have imposed a major obstacle to increasing crop production. - The apparent alteration of climates in places like Africa, Asia and other parts of the Northern hemisphere which may put significant restrictions on crop production. hey are cogent reasons to be seriously concerned about these matters. (author)

  12. Signal transduction by the major histocompatibility complex class I molecule

    DEFF Research Database (Denmark)

    Pedersen, A E; Skov, Svend; Bregenholt, S

    1999-01-01

    Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell...... and functioning, MHC-I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems....

  13. Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells.

    Science.gov (United States)

    Han, Li; Yu, Kefei

    2008-11-24

    Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4-null cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

  14. Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

    Science.gov (United States)

    De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferr