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Sample records for protein vitronectin increases

  1. Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall.

    PubMed

    Niculescu, F; Rus, H G; Poruţiu, D; Ghiurca, V; Vlaicu, R

    1989-08-01

    S-protein/vitronectin is a multifunctional glycoprotein interacting with both complement activation and coagulation pathways. Its presence was investigated in 5 femoral and 5 iliac atherosclerotic human arteries, obtained at surgery, by immunoelectron microscopy using an affinity purified rabbit IgG specific for human S-protein/vitronectin. The immunoelectron dense specific deposits were found in both intimal thickenings and fibrous plaques in association with elastic fibers, collagen bundles and cell debris in the vicinity of elastin. Cell debris embedded in the collagen matrix were S-protein/vitronectin negative. S-protein/vitronectin was also absent on intact cells, lipid droplets and cholesterol clefts. All cell debris, however, was positive for C5b-9 deposits suggesting that complement activation had occurred at these sites with or without S-protein/vitronectin interaction. S-protein/vitronectin may play a role in the arterial wall defence by restricting the extent of complement activation.

  2. Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance.

    PubMed

    Hallström, Teresia; Blom, Anna M; Zipfel, Peter F; Riesbeck, Kristian

    2009-08-15

    Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a K(D) of 0.4 microM for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84-108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.

  3. Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

    PubMed

    Bartra, Sara Schesser; Ding, Yi; Fujimoto, L Miya; Ring, Joshua G; Jain, Vishal; Ram, Sanjay; Marassi, Francesca M; Plano, Gregory V

    2015-11-01

    Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.

  4. Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin

    PubMed Central

    Bartra, Sara Schesser; Ding, Yi; Miya Fujimoto, L.; Ring, Joshua G.; Jain, Vishal; Ram, Sanjay; Marassi, Francesca M.

    2015-01-01

    Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin – a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs. PMID:26377177

  5. Vitronectin is A Critical Protein Adhesion Substrate for IL-4-INDUCED Foreign Body Giant Cell Formation

    PubMed Central

    McNally, Amy K.; Jones, Jacqueline A.; MacEwan, Sarah R.; Colton, Erica; Anderson, James M.

    2014-01-01

    An in vitro system of interleukin (IL)-4-induced foreign body giant cell (FBGC) formation was utilized to define the adhesion protein substrate(s) that promotes this aspect of the foreign body reaction on biomedical polymers. Human monocytes were cultured on cell culture polystyrene surfaces that had been pre-adsorbed with a synthetic arginine-glycine-aspartate (RGD) peptide previously found to support optimal FBGC formation, or with various concentrations of potential physiological protein substrates, i.e. complement C3bi, collagen types I or IV, fibrinogen, plasma fibronectin, fibroblast fibronectin, laminin, thrombospondin, vitronectin, or von Willebrand factor. Cultures were evaluated on days 0 (1.5 hr), 3, and 7 by May-Grünwald/Giemsa staining. Initial monocyte adhesion occurred on all adsorbed proteins. However, by day 7 of culture, only vitronectin was striking in its ability to support significant macrophage adhesion, development, and fusion leading to FBGC formation. Vitronectin supported high degrees of FBGC formation at an absorption concentration between 5 and 25 μg per ml. These findings suggest that adsorbed vitronectin is critical in the collective events that support and promote FBGC formation on biomedical polymers, and that the propensity for vitronectin adsorption may underlie the material surface chemistry dependency of FBGC formation. PMID:17994558

  6. Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells.

    PubMed

    Wu, J; Liu, W; Koenig, K; Idell, S; Broaddus, V C

    2000-11-01

    Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.

  7. Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

    PubMed

    Griffiths, Natalie J; Hill, Darryl J; Borodina, Elena; Sessions, Richard B; Devos, Nathalie I; Feron, Christiane M; Poolman, Jan T; Virji, Mumtaz

    2011-12-01

    Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

  8. Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

    PubMed

    Paulsson, Magnus; Singh, Birendra; Al-Jubair, Tamim; Su, Yu-Ching; Høiby, Niels; Riesbeck, Kristian

    2015-09-01

    Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells. We screened clinical isolates from the airways of CF patients and from the bloodstream of patients with bacteremia for binding of vitronectin. Two-dimensional SDS-PAGE and a proteomic approach were used to identify vitronectin-receptors in P. aeruginosa. P. aeruginosa from the airways of CF patients (n=27) bound more vitronectin than bacteremic isolates (n=15, p=0.025). Porin D (OprD) was identified as a vitronectin-binding protein. A P. aeruginosa oprD transposon insertion mutant had a decreased binding to soluble and immobilized vitronectin (p≤0.001). P. aeruginosa isolates obtained from CF patients significantly bound vitronectin. Porin D was defined as a novel P. aeruginosa vitronectin-receptor, and we postulate that the Porin D-dependent interaction with vitronectin may be important for colonization. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  9. Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Nagaoka, Masato; Kobayashi, Motohiro; Kawai, Chie; Mallanna, Sunil K.; Duncan, Stephen A.

    2015-01-01

    Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth—Holm—Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs. PMID:26308339

  10. Two classes of proteins and mRNAs in Lilium longiflorum L. indentified by human vitronectin probes.

    PubMed Central

    Wang, C S; Walling, L L; Gu, Y Q; Ware, C F; Lord, E M

    1994-01-01

    Vitronectin (VN) is a substrate adhesion molecule, an extracellular matrix glycoprotein that facilitates cell adhesion and cell movement in animals. We have reported the cross-reactivity of a 55-kD protein in plants with rabbit anti-human VN antibodies and the presence of VN-like sequences in plant genomes using a human VN cDNA probe. We have extended these studies by using human VN riboprobes to detect VN-like mRNAs in lily (Lilium longiflorum L.) and soybean. In both species, two mRNAs were detected. We have also identified a new cross-reactive protein (41 kD) using a different preparation of human VN antiserum. In lily roots five 41-kD isoforms were observed, whereas only three of these isoforms accumulated in leaves. Monospecific antibodies prepared against the plant proteins cross-reacted with the human VN protein and vice versa. We have purified the 41-kD protein using two-dimensional gel electrophoresis, and amino acid composition analysis indicates that it is similar in composition to human VN. PMID:7512737

  11. Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5.

    PubMed Central

    Boylan, A M; Sanan, D A; Sheppard, D; Broaddus, V C

    1995-01-01

    The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects. Images PMID:7560092

  12. Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

    PubMed Central

    Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva

    2016-01-01

    ABSTRACT Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal

  13. Localization of the domains of the Haemophilus ducreyi trimeric autotransporter DsrA involved in serum resistance and binding to the extracellular matrix proteins fibronectin and vitronectin.

    PubMed

    Leduc, Isabelle; Olsen, Bonnie; Elkins, Christopher

    2009-02-01

    Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi.

  14. Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

    PubMed

    Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva; Mohana-Borges, Ronaldo

    2016-11-01

    Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system.

  15. Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.

    PubMed

    Furutani, Yutaka; Kawasaki, Miwa; Matsuno, Hitomi; Mitsui, Sachiko; Mori, Kensaku; Yoshihara, Yoshihiro

    2012-11-09

    Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

  16. Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.

    PubMed

    Singh, Birendra; Jalalvand, Farshid; Mörgelin, Matthias; Zipfel, Peter; Blom, Anna M; Riesbeck, Kristian

    2011-07-01

    Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.

  17. A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*

    PubMed Central

    Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E.; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

    2005-01-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81—86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes. PMID:15905170

  18. Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.

    PubMed Central

    Waltz, D A; Natkin, L R; Fujita, R M; Wei, Y; Chapman, H A

    1997-01-01

    The urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis. PMID:9202057

  19. Reducing Timp3 or Vitronectin Ameliorates Disease Manifestations in CADASIL Mice

    PubMed Central

    Capone, Carmen; Cognat, Emmanuel; Ghezali, Lamia; Baron-Menguy, Céline; Aubin, Déborah; Mesnard, Laurent; Stöhr, Heidi; Domenga-Denier, Valérie; Nelson, Mark T.; Joutel, Anne

    2017-01-01

    Objective CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain (Notch3ECD) on the vessels. TIMP3 and vitronectin are 2 extracellular matrix proteins that abnormally accumulate in Notch3ECD-containing deposits on brain vessels of mice and patients with CADASIL. Herein, we investigated whether increased levels of TIMP3 and vitronectin are responsible for aspects of CADASIL disease phenotypes. Methods Timp3 and vitronectin expression were genetically reduced in TgNotch3R169C mice, a well-established preclinical model of CADASIL. A mouse overexpressing human TIMP3 (TgBAC-TIMP3) was developed. Disease-related phenotypes, including cerebral blood flow (CBF) deficits, white matter lesions, and Notch3ECD deposition, were evaluated between 6 and 20 months of age. Results CBF responses to neural activity (functional hyperemia), topical application of vasodilators, and decreases in blood pressure (CBF autoregulation) were similarly reduced in TgNotch3R169C and TgBAC-TIMP3 mice, and myogenic responses of brain arteries were likewise attenuated. These defects were rescued in TgNotch3R169C mice by haploinsufficiency of Timp3, although the number of white matter lesions was unaffected. In contrast, haploinsufficiency or loss of vitronectin in TgNotch3R169C mice ameliorated white matter lesions, although CBF responses were unchanged. Amelioration of cerebrovascular reactivity or white matter lesions in these mice was not associated with reduced Notch3ECD deposition in brain vessels. Interpretation Elevated levels of TIMP3 and vitronectin, acting downstream of Notch3ECD deposition, play a role in CADASIL, producing divergent influences on early CBF deficits and later white matter lesions. PMID:26648042

  20. Fibronectin and vitronectin promote human fetal osteoblast cell attachment and proliferation on nanoporous titanium surfaces

    PubMed Central

    Rivera-Chacon, D. M.; Alvarado-Velez, M.; Acevedo-Morantes, C.Y.; Singh, S.P.; Gultepe, E.; Nagesha, D.; Sridhar, S.; Ramirez-Vick, J.E.

    2013-01-01

    Improvements in osteoconduction of implant biomaterials require focusing on the bone-implant interface, which is a complex multifactorial system. Surface topography of implants plays a crucial role at this interface. Nanostructured surfaces have been shown to promote serum protein adsorption and osteoblast adhesion when compared to microstructured surfaces for bone-implant materials. We studied the influence of the serum proteins fibronectin and vitronectin on the attachment and proliferation of osteoblasts onto nanostructured titania surfaces. Human fetal osteoblastic cells hFOB 1.19 were used as model osteoblasts and were grown on nanoporous TiO2 templates, using Ti6Al4V and commercially pure Ti substrates as controls. Results show a significant increase in cell proliferation on nanoporous TiO2 over flat substrates. Initial cell attachment data exhibited a significant effect by either fibronectin or vitronectin on cell adhesion at the surface of any of the tested materials. In addition, the extent of cell adhesion was significantly different between the nanoporous TiO2 and both Ti6Al4V and commercially pure Ti substrates, with the first showing the highest surface coverage. There was no significant difference on osteoblast attachment or proliferation between the presence of fibronectin or vitronectin using any of the material substrates. Taken together, these results suggest that the increase in osteoblast attachment and proliferation shown on the nanoporous TiO2 is due to an increase in the adsorption of fibronectin and vitronectin because of the higher surface area and to an enhanced protein unfolding, which allows access to osteoblast binding motifs within these proteins. PMID:23858975

  1. A chimeric vitronectin: IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells.

    PubMed

    Manton, Kerry J; Richards, Sean; Van Lonkhuyzen, Derek; Cormack, Luke; Leavesley, David; Upton, Zee

    2010-09-01

    The therapeutic use of human embryonic stem (hES) cells is severely limited by safety concerns regarding their culture in media containing animal-derived or nondefined factors and on animal-derived feeder cells. Thus, there is a pressing need to develop culture techniques that are xeno-free, fully defined, and synthetic. Our laboratory has discovered that insulin-like growth factor (IGF) and vitronectin (VN) bind to each other resulting in synergistic short-term functional effects in several cell types, including keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric VN:IGF-I protein that functionally mimics the effects obtained upon binding of IGF-I to VN. The aim of the current study was to determine whether hES cells can be serially propagated in feeder-cell-free and serum-free conditions using medium containing our novel chimeric VN:IGF-I protein. Here we demonstrate that hES cells can be serially propagated and retain their undifferentiated state in vitro for up to 35 passages in our feeder-cell-free, serum-free, chemically defined media. We have utilized real-time polymerase chain reaction (PCR), immunofluorescence, and fluorescence-activated cell sorter (FACS) analysis to show that the hES cells have maintained an undifferentiated phenotype. In vitro differentiation assays demonstrated that the hES cells retain their pluripotent potential and the karyotype of the hES cells remains unchanged. This study demonstrates that the novel, fully defined, synthetic VN:IGF-I chimera-containing medium described herein is a viable alternative to media containing serum, and that in conjunction with laminin-coated plates facilitates feeder-cell-free and serum-free growth of hES.

  2. Vitronectin Expression in the Airways of Subjects with Asthma and Chronic Obstructive Pulmonary Disease

    PubMed Central

    Salazar-Peláez, Lina M.; Abraham, Thomas; Herrera, Ana M.; Correa, Mario A.; Ortega, Jorge E.; Paré, Peter D.; Seow, Chun Y.

    2015-01-01

    Vitronectin, a multifunctional glycoprotein, is involved in coagulation, inhibition of the formation of the membrane attack complex (MAC), cell adhesion and migration, wound healing, and tissue remodeling. The primary cellular source of vitronectin is hepatocytes; it is not known whether resident cells of airways produce vitronectin, even though the glycoprotein has been found in exhaled breath condensate and bronchoalveolar lavage from healthy subjects and patients with interstitial lung disease. It is also not known whether vitronectin expression is altered in subjects with asthma and COPD. In this study, bronchial tissue from 7 asthmatic, 10 COPD and 14 control subjects was obtained at autopsy and analyzed by immunohistochemistry to determine the percent area of submucosal glands occupied by vitronectin. In a separate set of experiments, quantitative colocalization analysis was performed on tracheobronchial tissue sections obtained from donor lungs (6 asthmatics, 4 COPD and 7 controls). Vitronectin RNA and protein expressions in bronchial surface epithelium were examined in 12 subjects who undertook diagnostic bronchoscopy. Vitronectin was found in the tracheobronchial epithelium from asthmatic, COPD, and control subjects, although its expression was significantly lower in the asthmatic group. Colocalization analysis of 3D confocal images indicates that vitronectin is expressed in the glandular serous epithelial cells and in respiratory surface epithelial cells other than goblet cells. Expression of the 65-kDa vitronectin isoform was lower in bronchial surface epithelium from the diseased subjects. The cause for the decreased vitronectin expression in asthma is not clear, however, the reduced concentration of vitronectin in the epithelial/submucosal layer of airways may be linked to airway remodeling. PMID:25768308

  3. Induction of carcinoma cell migration on vitronectin by NF-kappa B-dependent gene expression.

    PubMed Central

    Yebra, M; Filardo, E J; Bayna, E M; Kawahara, E; Becker, J C; Cheresh, D A

    1995-01-01

    Integrin alpha v beta 5 promotes FG carcinoma cell adhesion to vitronectin yet requires protein kinase C (PKC) activation for migration on this ligand. Here we report that this PKC-dependent cell motility event requires NF-kappaB-dependent transcription. Specifically, a component within nuclear extracts prepared from PKC-stimulated FG cells exhibited a significant increase in binding activity to a synthetic oligonucleotide containing a consensus kappa B sequence. These nuclear DNA-binding complexes were shown to be comprised of p65 and p50 NF-kappaB/rel family members and appeared functionally active because they promoted transcription of a reporter construct containing a kappa B site. The NF-kappa B activation event was directly linked to the alpha v beta 5 motility response because the NF-kappa B-binding oligonucleotide, when introduced into FG cells, inhibited cell migration on vitronectin but not on collagen and had no effect on cell adhesion to either ligand. These results suggest that the detected DNA-binding complexes interact with kappa B transcriptional elements to regulate gene expression required for alpha v beta 5-dependent cell motility on vitronectin. Images PMID:7579698

  4. Binding of vitronectin and Factor H to Hic contributes to immune evasion of Streptococcus pneumoniae serotype 3.

    PubMed

    Kohler, Sylvia; Hallström, Teresia; Singh, Birendra; Riesbeck, Kristian; Spartà, Giuseppina; Zipfel, Peter F; Hammerschmidt, Sven

    2015-01-01

    Streptococcus pneumoniae serotype 3 strains are highly resistant to opsonophagocytosis due to recruitment of the complement inhibitor Factor H via Hic, a member of the pneumococcal surface protein C (PspC) family. In this study, we demonstrated that Hic also interacts with vitronectin, a fluid-phase regulator involved in haemostasis, angiogenesis, and the terminal complement cascade as well as a component of the extracellular matrix. Blocking of Hic by specific antiserum or genetic deletion significantly reduced pneumococcal binding to soluble and immobilised vitronectin and to Factor H, respectively. In parallel, ectopic expression of Hic on the surface of Lactococcus lactis conferred binding to soluble and immobilised vitronectin as well as Factor H. Molecular analyses with truncated Hic fragments narrowed down the vitronectin-binding site to the central core of Hic (aa 151-201). This vitronectin-binding region is separate from that of Factor H, which binds to the N-terminus of Hic (aa 38-92). Binding of pneumococcal Hic was localised to the C-terminal heparin-binding domain (HBD3) of vitronectin. However, an N-terminal region to HBD3 was further involved in Hic-binding to immobilised vitronectin. Finally, vitronectin bound to Hic was functionally active and inhibited formation of the terminal complement complex. In conclusion, Hic interacts with vitronectin and simultaneously with Factor H, and both human proteins may contribute to colonisation and invasive disease caused by serotype 3 pneumococci.

  5. New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1.vitronectin complex.

    PubMed

    Podor, T J; Shaughnessy, S G; Blackburn, M N; Peterson, C B

    2000-08-18

    Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.

  6. Vitronectin and dermcidin serum levels predict the metastatic progression of AJCC I–II early‐stage melanoma

    PubMed Central

    Ortega‐Martínez, Idoia; Gardeazabal, Jesús; Erramuzpe, Asier; Sanchez‐Diez, Ana; Cortés, Jesús; García‐Vázquez, María D.; Pérez‐Yarza, Gorka; Izu, Rosa; Luís Díaz‐Ramón, Jose; de la Fuente, Ildefonso M.; Asumendi, Aintzane

    2016-01-01

    Like many cancers, an early diagnosis of melanoma is fundamental to ensure a good prognosis, although an important proportion of stage I–II patients may still develop metastasis during follow‐up. The aim of this work was to discover serum biomarkers in patients diagnosed with primary melanoma that identify those at a high risk of developing metastasis during the follow‐up period. Proteomic and mass spectrophotometry analysis was performed on serum obtained from patients who developed metastasis during the first years after surgery for primary tumors and compared with that from patients who remained disease‐free for more than 10 years after surgery. Five proteins were selected for validation as prognostic factors in 348 melanoma patients and 100 controls by ELISA: serum amyloid A and clusterin; immune system proteins; the cell adhesion molecules plakoglobin and vitronectin and the antimicrobial protein dermcidin. Compared to healthy controls, melanoma patients have high serum levels of these proteins at the moment of melanoma diagnosis, although the specific values were not related to the histopathological stage of the tumors. However, an analysis based on classification together with multivariate statistics showed that tumor stage, vitronectin and dermcidin levels were associated with the metastatic progression of patients with early‐stage melanoma. Although melanoma patients have increased serum dermcidin levels, the REPTree classifier showed that levels of dermcidin <2.98 μg/ml predict metastasis in AJCC stage II patients. These data suggest that vitronectin and dermcidin are potent biomarkers of prognosis, which may help to improve the personalized medical care of melanoma patients and their survival. PMID:27216146

  7. Measurement of interaction force between nanoarrayed integrin {alpha}{sub v}{beta}{sub 3} and immobilized vitronectin on the cantilever tip

    SciTech Connect

    Lee, Minsu; Yang, Hyun-Kyu; Park, Keun-Hyung; Kang, Dong-Ku; Chang, Soo-Ik Kang, In-Cheol

    2007-11-03

    Protein nanoarrays containing integrin {alpha}{sub v}{beta}{sub 3} or BSA were fabricated on ProLinker{sup TM}-coated Au surface by dip-pen nanolithography (DPN). An atomic force microscope (AFM) tip coated with ProLinker{sup TM} was modified by vitronectin. We measured the interaction force between nanoarrayed integrin {alpha}{sub v}{beta}{sub 3} or BSA and immobilized vitronectin on the cantilever tip by employing tethering-unbinding method. The unbinding force between integrin {alpha}{sub v}{beta}{sub 3} and vitronectin (1087 {+-} 62 pN) was much higher than that of between BSA and vitronectin (643 {+-} 74 pN). These results demonstrate that one can distinguish a specific protein interaction from non-specific interactions by means of force measurement on the molecular interactions between the nanoarrayed protein and its interacting protein on the AFM tip.

  8. Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif

    PubMed Central

    Kraiczy, Peter; Hammerschmidt, Sven; Skerka, Christine; Zipfel, Peter F.; Riesbeck, Kristian

    2016-01-01

    Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352–374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response. PMID:26808444

  9. Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif.

    PubMed

    Hallström, Teresia; Singh, Birendra; Kraiczy, Peter; Hammerschmidt, Sven; Skerka, Christine; Zipfel, Peter F; Riesbeck, Kristian

    2016-01-01

    Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352-374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response.

  10. Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

    PubMed

    Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

    2015-01-01

    The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

  11. Glioblastoma expression of vitronectin and the alpha v beta 3 integrin. Adhesion mechanism for transformed glial cells.

    PubMed Central

    Gladson, C L; Cheresh, D A

    1991-01-01

    Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain. Images PMID:1721625

  12. Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Plopper, George E.

    2004-01-01

    The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin>collagen I≥collagen IV≥vitronectin>laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells. PMID:15123885

  13. The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells.

    PubMed

    Martí-Pàmies, Ingrid; Cañes, Laia; Alonso, Judith; Rodríguez, Cristina; Martínez-González, José

    2017-10-01

    The nuclear receptor NOR-1 (NR4A3) has recently been involved in the regulation of extracellular matrix (ECM) proteins associated with neointimal thickening and the vascular control of hemostasis. We sought to find as-yet unidentified NOR-1 target genes in human vascular smooth muscle cells (VSMCs). An in silico analysis identified putative NOR-1 response elements in the proximal promoter region of several genes encoding for ECM proteins, including vitronectin (VTN). Lentiviral overexpression of NOR-1 strongly increased VTN mRNA and protein levels, whereas NOR-1 silencing significantly reduced VTN expression. Deletion and site-directed mutagenesis studies, as well as EMSA and chromatin immunoprecipitation, identified the NBRE(-202/-195) site in the VTN promoter as an essential element for NOR-1 responsiveness. Furthermore, NOR-1 and VTN colocalized in VSMCs in human atherosclerotic lesions. VTN levels were increased in cell supernatants from VSMCs that overexpress NOR-1. Cell supernatants from VSMCs overexpressing NOR-1 induced cell migration to a greater extent than supernatants from control cells, and this effect was attenuated when cell supernatants were preincubated with anti-VTN blocking antibodies or VTN was silenced in supernatant-generating cells. These results indicate that VTN is a target of NOR-1 and suggest that this multifunctional glycoprotein may participate in vascular responses mediated by this nuclear receptor.-Martí-Pàmies, I., Cañes, L., Alonso, J., Rodríguez, C., Martínez-González, J. The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells. © FASEB.

  14. Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

    PubMed Central

    Stuart, Christopher H; Riley, Kathryn R; Boyacioglu, Olcay; Herpai, Denise M; Debinski, Waldemar; Qasem, Shadi; Marini, Frank C; Colyer, Christa L.; Gmeiner, William H

    2016-01-01

    Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10−6mol/l) relative to uncoated plates (2.4 × 10−6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10−6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC. PMID:27845768

  15. Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.

    PubMed

    Stuart, Christopher H; Riley, Kathryn R; Boyacioglu, Olcay; Herpai, Denise M; Debinski, Waldemar; Qasem, Shadi; Marini, Frank C; Colyer, Christa L; Gmeiner, William H

    2016-11-15

    Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5(°)C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10(-6)mol/l) relative to uncoated plates (2.4 × 10(-6) mol/l), or plates coated with the related protein fibronectin (2.1 × 10(-6) mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

  16. Crocidolite Induces Prostaglandin I2 Release Mediated by Vitronectin Receptor and Cyclooxygenase-2 in Lung Cells

    PubMed Central

    Roberts, Kevan

    2010-01-01

    Interstitial lung disease (ILD) produces disruption of alveolar walls with loss of functionality and scar tissue accumulation. Asbestosis is the ILD produced by the inhalation of asbestos fibers. This study attempts to elucidate the role of lung epithelial cells in the generation of asbestos-induced ILD. When exposed to crocidolite LA-4 cells had a decrease in viability and an increase in the release of lactate dehydrogenase (LDH) and 6-keto PGF1α, a PGI2 metabolite. PGI2 release was mediated by cyclo-oxygenase-2 (COX-2) and vitronectin receptor (VNR). When LA-4 cells were treated with VNR inhibitors, either RGD (Arg-Gly-Asp) peptide or VNR blocking antibody, a statistically significant decrease in PGI2 metabolite production was observed, but crocidolite-induced cytotoxicity was not prevented. These findings propose that crocidolite is coated by an RGD protein and binds VNR-inducing COX-2 expression and PGI2 release. Moreover, when LA-4 cells were exposed to crocidolite in the presence of reduced serum culture media, PGI2 production was prevented, and when bronchoalveolar lavage fluid (BALF) was added, PGI2 production was rescued. Cytotoxicity did not occur, either in reduced serum culture media or when BALF was added. In conclusion, crocidolite requires the presence of an RGD protein coating the fibers to induce inflammation (PGI2 production) and crocidolite alone cannot induce cytotoxicity in lung cells. PMID:20155273

  17. Pathogenic Rickettsia Species Acquire Vitronectin from Human Serum to Promote Resistance to Complement-mediated Killing

    PubMed Central

    Riley, Sean P.; Patterson, Jennifer L.; Nava, Samantha; Martinez, Juan J.

    2014-01-01

    SUMMARY Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer-membrane of serum sensitive E. coli. Adr1 is an integral outer-membrane protein whose structure is predicted to contain eight membrane-embedded β-strands and four “loop” regions that are exposed to extracellular milieu. Site-directed mutagenesis of Adr1 revealed that at least two predicted “loop” regions are required to mediate resistance to complement-mediated killing and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens. PMID:24286496

  18. The Vitronectin Receptor and its Associated CD47 Molecule Mediates Proinflammatory Cytokine Synthesis in Human Monocytes by Interaction with Soluble CD23

    PubMed Central

    Hermann, P.; Armant, M.; Brown, E.; Rubio, M.; Ishihara, H.; Ulrich, D.; Caspary, R.G.; Lindberg, F.P.; Armitage, R.; Maliszewski, C.; Delespesse, G.; Sarfati, M.

    1999-01-01

    The vitronectin receptor, αvβ3 integrin, plays an important role in tumor cell invasion, angiogenesis, and phagocytosis of apoptotic cells. CD47, a member of the multispan transmembrane receptor family, physically and functionally associates with vitronectin receptor (VnR). Although vitronectin (Vn) is not a ligand of CD47, anti-CD47 and β3 mAbs suppress Vn, but not fibronectin (Fn) binding and function. Here, we show that anti-CD47, anti-β3 mAb and Vn, but not Fn, inhibit sCD23-mediated proinflammatory function (TNF-α, IL-12, and IFN-γ release). Surprisingly, anti-CD47 and β3 mAbs do not block sCD23 binding to αv+β3+ T cell lines, whereas Vn and an αv mAb (clone AMF7) do inhibit sCD23 binding, suggesting the VnR complex may be a functional receptor for sCD23. sCD23 directly binds αv+β3+/CD47− cell lines, but coexpression of CD47 increases binding. Moreover, sCD23 binds purified αv protein and a single human αv chain CHO transfectant. We conclude that the VnR and its associated CD47 molecule may function as a novel receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response. PMID:10037797

  19. Vitronectin is not essential for normal mammalian development and fertility.

    PubMed Central

    Zheng, X; Saunders, T L; Camper, S A; Samuelson, L C; Ginsburg, D

    1995-01-01

    Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618914

  20. Vitronectin-binding staphylococci enhance surface-associated complement activation.

    PubMed Central

    Lundberg, F; Lea, T; Ljungh, A

    1997-01-01

    Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn. PMID:9038294

  1. The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner

    PubMed Central

    Fish, Abigail I.; Riley, Sean P.; Singh, Birendra; Riesbeck, Kristian; Martinez, Juan J.

    2017-01-01

    Spotted fever group (SFG) Rickettsia species are inoculated into the mammalian bloodstream by hematophagous arthropods. Once in the bloodstream and during dissemination, the survival of these pathogens is dependent upon the ability of these bacteria to evade serum-borne host defenses until a proper cellular host is reached. Rickettsia conorii expresses an outer membrane protein, Adr1, which binds the complement inhibitory protein vitronectin to promote resistance to the anti-bacterial effects of the terminal complement complex. Adr1 is predicted to consist of 8 transmembrane beta sheets that form a membrane-spanning barrel with 4 peptide loops exposed to the extracellular environment. We previously demonstrated that Adr1 derivatives containing either loop 3 or 4 are sufficient to bind Vn and mediate resistance to serum killing when expressed at the outer-membrane of E. coli. By expressing R. conorii Adr1 on the surface of non-pathogenic E. coli, we demonstrate that the interaction between Adr1 and vitronectin is salt-sensitive and cannot be interrupted by addition of heparin. Additionally, we utilized vitroenctin-derived peptides to map the minimal Adr1/vitronectin interaction to the C-terminal region of vitronectin. Furthermore, we demonstrate that specific charged amino acid residues located within loops 3 and 4 of Adr1 are critical for mediating resistance to complement-mediated killing. Interestingly, Adr1 mutants that were no longer sufficient to mediate resistance to serum killing still retained the ability to bind to Vn, suggesting that Adr1-Vn interactions responsible for resistance to serum killing are more complex than originally hypothesized. In summary, elucidation of the mechanisms governing Adr1-Vn binding will be useful to specifically target this protein-protein interaction for therapeutic intervention. PMID:28299286

  2. The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner.

    PubMed

    Fish, Abigail I; Riley, Sean P; Singh, Birendra; Riesbeck, Kristian; Martinez, Juan J

    2017-01-01

    Spotted fever group (SFG) Rickettsia species are inoculated into the mammalian bloodstream by hematophagous arthropods. Once in the bloodstream and during dissemination, the survival of these pathogens is dependent upon the ability of these bacteria to evade serum-borne host defenses until a proper cellular host is reached. Rickettsia conorii expresses an outer membrane protein, Adr1, which binds the complement inhibitory protein vitronectin to promote resistance to the anti-bacterial effects of the terminal complement complex. Adr1 is predicted to consist of 8 transmembrane beta sheets that form a membrane-spanning barrel with 4 peptide loops exposed to the extracellular environment. We previously demonstrated that Adr1 derivatives containing either loop 3 or 4 are sufficient to bind Vn and mediate resistance to serum killing when expressed at the outer-membrane of E. coli. By expressing R. conorii Adr1 on the surface of non-pathogenic E. coli, we demonstrate that the interaction between Adr1 and vitronectin is salt-sensitive and cannot be interrupted by addition of heparin. Additionally, we utilized vitroenctin-derived peptides to map the minimal Adr1/vitronectin interaction to the C-terminal region of vitronectin. Furthermore, we demonstrate that specific charged amino acid residues located within loops 3 and 4 of Adr1 are critical for mediating resistance to complement-mediated killing. Interestingly, Adr1 mutants that were no longer sufficient to mediate resistance to serum killing still retained the ability to bind to Vn, suggesting that Adr1-Vn interactions responsible for resistance to serum killing are more complex than originally hypothesized. In summary, elucidation of the mechanisms governing Adr1-Vn binding will be useful to specifically target this protein-protein interaction for therapeutic intervention.

  3. Control of adhesion of human induced pluripotent stem cells to plasma-patterned polydimethylsiloxane coated with vitronectin and γ-globulin.

    PubMed

    Yamada, Ryotaro; Hattori, Koji; Tachikawa, Saoko; Tagaya, Motohiro; Sasaki, Toru; Sugiura, Shinji; Kanamori, Toshiyuki; Ohnuma, Kiyoshi

    2014-09-01

    Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices.

  4. uPA Binding to PAI-1 Induces Corneal Myofibroblast Differentiation on Vitronectin

    PubMed Central

    Wang, Lingyan; Ly, Christine M.; Ko, Chun-Ying; Meyers, Erin E.; Lawrence, Daniel A.; Bernstein, Audrey M.

    2012-01-01

    Purpose. Vitronectin (VN) in provisional extracellular matrix (ECM) promotes cell migration. Fibrotic ECM also includes VN and, paradoxically, strongly adherent myofibroblasts (Mfs). Because fibrotic Mfs secrete elevated amounts of urokinase plasminogen activator (uPA), we tested whether increased extracellular uPA promotes the persistence of Mfs on VN. Methods. Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free medium on VN or collagen (CL) with 1ng/mL transforming growth factor β1 (TGFβ1). Adherent cells were quantified using crystal violet. Protein expression was measured by Western blotting and flow cytometry. Transfection of short interfering RNAs was performed by nucleofection. Mfs were identified by α-smooth muscle actin (α-SMA) stress fibers. Plasminogen activator inhibitor (PAI-1) levels were quantified by ELISA. Results. TGFβ1-treated HCFs secreted PAI-1 (0.5uM) that bound to VN, competing with αvβ3/αvβ5 integrin/VN binding, thus promoting cell detachment from VN. However, addition of uPA to cells on VN increased Mf differentiation (9.7-fold), cell-adhesion (2.2-fold), and binding by the VN integrins αvβ3 and -β5 (2.2-fold). Plasmin activity was not involved in promoting these changes, as treatment with the plasmin inhibitor aprotinin had no effect. A dominant negative PAI-1 mutant (PAI-1R) that binds to VN but does not inhibit uPA prevented the increase in uPA-stimulated cell adhesion and reduced uPA-stimulated integrin αvβ3/αvβ5 binding to VN by 73%. Conclusions. uPA induction of TGFβ1-dependent Mf differentiation on VN supports the hypothesis that elevated secretion of uPA in fibrotic tissue may promote cell adhesion and the persistence of Mfs. By blocking uPA-stimulated cell adhesion, PAI-1R may be a useful agent in combating corneal scarring. PMID:22700714

  5. Propolis modulates vitronectin, laminin, and heparan sulfate/heparin expression during experimental burn healing.

    PubMed

    Olczyk, Paweł; Komosińska-Vassev, Katarzyna; Winsz-Szczotka, Katarzyna; Koźma, Ewa M; Wisowski, Grzegorz; Stojko, Jerzy; Klimek, Katarzyna; Olczyk, Krystyna

    2012-11-01

    This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. VN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis. Propolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD. The beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management.

  6. Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation.

    PubMed

    Lygoe, Kate A; Wall, Ivan; Stephens, Philip; Lewis, Mark P

    2007-11-01

    The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient-matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Both the HOF and HDF cell types underwent TGF-beta1 (transforming growth factor-beta1)-induced myofibroblastic differentiation [upregulation of the expression of alpha-sma (alpha-smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of alpha-sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits alpha 5 (fibronectin) or alpha v (vitronectin) were used to determine whether the effects of TGF-beta1 were regulated via integrin signalling pathways. alpha-sma expression in both HOFs and HDFs was down-regulated by antibodies against both alpha 5 and alpha v. Functionally, TGF-beta1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF-beta1 (P<0.05). When TGF-beta1-stimulated cells were incubated with blocking antibodies against alpha 5 and alpha v, gel contraction was decreased to that of non-stimulated cells; however, blocking alpha v or alpha 5 could not restore cellular migration in both HOFs and HDFs. Despite intrinsic differences in their basal state, the cellular events associated with TGF-beta1-induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up-regulation of alpha-sma expression and increases in collagen gel contraction are vitronectin- and fibronectin-receptor-dependent processes, whereas wound re-population is not.

  7. Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration

    PubMed Central

    1991-01-01

    Transient increases in cytosolic free calcium concentration, [Ca2+]i, appear to be required for the migration of human neutrophils on poly-D- lysine-coated glass in the presence of dilute serum (Marks, P. W., and F. R. Maxfield. 1990. J. Cell Biol. 110:43-52). In contrast, no requirement for [Ca2+]i transients exists when neutrophils migrate on albumin-coated glass in the absence of serum. To determine the mechanism that necessitates [Ca2+]i transients on poly-D-lysine in the presence of serum, migration was examined on substrates consisting of purified adhesive glycoproteins. In the absence of external Ca2+, a treatment which causes the cessation of [Ca2+]i transients, migration on fibronectin (fn) and vitronectin (vn) was significantly inhibited. Migration was also inhibited in Ca2(+)-buffered cells on these substrates, indicating that this effect was the result of an alteration of [Ca2+]i. In the absence of external Ca2+, the inhibition of migration on fn or vn was more pronounced when soluble fn or vn was added to cells migrating on these substrates. This effect of soluble adhesive glycoprotein was specific: in the absence of external Ca2+, soluble fn did not affect the migration of cells on vn, and soluble vn did not affect the migration on fn. No additional inhibition of migration was observed in Ca2(+)-buffered cells with the addition of soluble adhesive glycoprotein. These data indicate that [Ca2+]i transients are involved in continued migration of human neutrophils on fn or vn, proteins which are part of the extracellular matrix that neutrophils encounter in vivo. PMID:1702443

  8. Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor

    PubMed Central

    1990-01-01

    Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI- 1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin- coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor. PMID:1697297

  9. Selection of mutations for increased protein stability.

    PubMed

    van den Burg, Bertus; Eijsink, Vincent G H

    2002-08-01

    There are many ways to select mutations that increase the stability of proteins, including rational design, functional screening of randomly generated mutant libraries, and comparison of naturally occurring homologous proteins. The protein engineer's toolbox is expanding and the number of successful examples of engineered protein stability is increasing. Still, the selection of thermostable mutations is not a standard process. Selection is complicated by lack of knowledge of the process that leads to thermal inactivation and by the fact that proteins employ a large variety of structural tricks to achieve stability.

  10. Increased protein intake in military special operations.

    PubMed

    Ferrando, Arny A

    2013-11-01

    Special operations are so designated for the specialized military missions they address. As a result, special operations present some unique metabolic challenges. In particular, soldiers often operate in a negative energy balance in stressful and demanding conditions with little opportunity for rest or recovery. In this framework, findings inferred from the performance literature suggest that increased protein intake may be beneficial. In particular, increased protein intake during negative caloric balance maintains lean body mass and blood glucose production. The addition of protein to mixed macronutrient supplements is beneficial for muscle endurance and power endpoints, and the use of amino acids improves gross and fine motor skills. Increasing protein intake during periods of intense training and/or metabolic demand improves subsequent performance, improves muscular recovery, and reduces symptoms of psychological stress. Consumption of protein before sleep confers the anabolic responses required for the maintenance of lean mass and muscle recovery. A maximal response in muscle protein synthesis is achieved with the consumption of 20-25 g of protein alone. However, higher protein intakes in the context of mixed-nutrient ingestion also confer anabolic benefits by reducing protein breakdown. Restricted rations issued to special operators provide less than the RDA for protein ( ∼ 0.6 g/kg), and these soldiers often rely on commercial products to augment their rations. The provision of reasonable alternatives and/or certification of approved supplements by the U.S. Department of Defense would be prudent.

  11. Hybrid vitronectin-mimicking polycaprolactone scaffolds for human retinal progenitor cell differentiation and transplantation.

    PubMed

    Lawley, Elodie; Baranov, Petr; Young, Michael

    2015-01-01

    Many advances have been made in an attempt to treat retinal degenerative diseases, such as age-related macular degeneration and retinitis pigmentosa. The irreversible loss of photoreceptors is common to both, and currently no restorative clinical treatment exists. It has been shown that retinal progenitor and photoreceptor precursor cell transplantation can rescue the retinal structure and function. Importantly, retinal progenitor cells can be collected from the developing neural retina with further expansion and additional modification in vitro, and the delivery into the degenerative host can be performed as a single-cell suspension injection or as a complex graft transplantation. Previously, we have described several polymer scaffolds for culture and transplantation of retinal progenitor cells of both mouse and human origin. This tissue engineering strategy increases donor cell survival and integration. We have also shown that biodegradable poly(ɛ-caprolactone) induces mature photoreceptor differentiation from human retinal progenitor cells. However, poor adhesive properties limit its use, and therefore it requires additional surface modification. The aim of this work was to study vitronectin-mimicking oligopeptides (Synthemax II-SC) poly(ɛ-caprolactone) films and their effects on human retinal progenitor cell adhesion, proliferation, and differentiation. Here, we show that the incorporation of vitronectin-mimicking oligopeptide into poly(ɛ-caprolactone) leads to dose-dependent increases in cell adhesion; the optimum dose identified as 30 µg/ml. Inhibition of human retinal progenitor cells proliferation was seen on poly(ɛ-caprolactone) and was maintained with the hybrid scaffold. This has been shown to be beneficial for driving cell differentiation. Additionally, we observed equal expression of Nrl, rhodopsin, recoverin, and rod outer membrane 1 after differentiation on the hybrid scaffold as compared to the standard fibronectin coating of poly

  12. Detection of vitronectin mRNA in tissues and cells of the mouse.

    PubMed Central

    Seiffert, D; Keeton, M; Eguchi, Y; Sawdey, M; Loskutoff, D J

    1991-01-01

    Mouse vitronectin (Vn) was isolated from serum by heparin affinity chromatography. The purified protein (Mr 71,000) supported adhesion of mouse and human cells in an Arg-Gly-Asp-dependent manner and bound to type 1 plasminogen activator inhibitor with kinetics similar to those observed using human and bovine Vn. To further characterize murine Vn and its biosynthesis in vivo, a mouse Vn cDNA was isolated from a liver cDNA library. The amino acid sequence of mouse Vn was deduced from the cDNA and was aligned with that of human Vn. Based on this alignment, mouse Vn was inferred to be 457 amino acids long and to have extensive (82%) homology with human Vn. Northern blot hybridization analysis of RNA from mouse tissues, using the mouse Vn cDNA as a hybridization probe, revealed the presence of a single transcript of 1.7 kilobases in mouse liver. Vn mRNA was not detectable in heart, lung, kidney, spleen, muscle, brain, thymus, testes, uterus, skin, adipose tissue, and aorta. The cellular localization of liver Vn mRNA was studied by in situ hybridization. Strong staining was observed only in hepatocytes, suggesting that these cells are the primary source of Vn in vivo. Images PMID:1719529

  13. Can protein levels be economically increased?

    USDA-ARS?s Scientific Manuscript database

    One result from the 2010 hard red winter wheat harvest was an increase of discussions on protein values across the southern great plains. The crop garnered relatively low protein values for several reasons, many of which were directly related to the weather patterns and environmental conditions. T...

  14. Increasing the protein content of ice cream.

    PubMed

    Patel, M R; Baer, R J; Acharya, M R

    2006-05-01

    Vanilla ice cream was made with a mix composition of 10.5% milk fat, 10.5% milk SNF, 12% beet sugar, and 4% corn syrup solids. None of the batches made contained stabilizer or emulsifier. The control (treatment 1) contained 3.78% protein. Treatments 2 and 5 contained 30% more protein, treatments 3 and 6 contained 60% more protein, and treatments 4 and 7 contained 90% more protein compared with treatment 1 by addition of whey protein concentrate or milk protein concentrate powders, respectively. In all treatments, levels of milk fat, milk SNF, beet sugar, and corn syrup solids were kept constant at 37% total solids. Mix protein content for treatment 1 was 3.78%, treatment 2 was 4.90%, treatment 5 was 4.91%, treatments 3 and 6 were 6.05%, and treatments 4 and 7 were 7.18%. This represented a 29.89, 60.05, 89.95, 29.63, 60.05, and 89.95% increase in protein for treatment 2 through treatment 7 compared with treatment 1, respectively. Milk protein level influenced ice crystal size; with increased protein, the ice crystal size was favorably reduced in treatments 2, 4, and 5 and was similar in treatments 3, 6, and 7 compared with treatment 1. At 1 wk postmanufacture, overall texture acceptance for all treatments was more desirable compared with treatment 1. When evaluating all parameters, treatment 2 with added whey protein concentrate and treatments 5 and 6 with added milk protein concentrate were similar or improved compared with treatment 1. It is possible to produce acceptable ice cream with higher levels of protein.

  15. Antagonists of IGF:Vitronectin Interactions Inhibit IGF-I-Induced Breast Cancer Cell Functions.

    PubMed

    Kashyap, Abhishek S; Shooter, Gary K; Shokoohmand, Ali; McGovern, Jacqui; Sivaramakrishnan, Manaswini; Croll, Tristan I; Cane, Gaëlle; Leavesley, David I; Söderberg, Ola; Upton, Zee; Hollier, Brett G

    2016-07-01

    We provide proof-of-concept evidence for a new class of therapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes with IGF-binding proteins (IGFBP) and the ECM protein vitronectin (VN), and stimulates the survival, migration and invasion of breast cancer cells. For the first time we provide physical evidence for IGFBP-3:VN interactions in breast cancer patient tissues; these interactions were predominantly localized to tumor cell clusters and in stroma surrounding tumor cells. We show that disruption of IGF-I:IGFBP:VN complexes with L(27)-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and proliferation in two- and three-dimensional assay systems. Peptide arrays screened to identify regions critical for the IGFBP-3/-5:VN and IGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-II binds VN through the hemopexin-2 domain, and VN binds IGFBP-3 at residues not involved in the binding of IGF-I to IGFBP-3. IGFBP-interacting VN peptides identified from these peptide arrays disrupted the IGF-I:IGFBP:VN complex, impeded the growth of primary tumor-like spheroids and, more importantly, inhibited the invasion of metastatic breast cancer cells in 3D assay systems. These studies provide first-in-field evidence for the utility of small peptides in antagonizing GF:ECM-mediated biologic functions and present data demonstrating the potential of these peptide antagonists as novel therapeutics. Mol Cancer Ther; 15(7); 1602-13. ©2016 AACR. ©2016 American Association for Cancer Research.

  16. Increased flexibility decreases antifreeze protein activity.

    PubMed

    Patel, Shruti N; Graether, Steffen P

    2010-12-01

    Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface. Copyright © 2010 The Protein Society.

  17. Protein ingestion increases myofibrillar protein synthesis after concurrent exercise.

    PubMed

    Camera, Donny M; West, Daniel W D; Phillips, Stuart M; Rerecich, Tracy; Stellingwerff, Trent; Hawley, John A; Coffey, Vernon G

    2015-01-01

    We determined the effect of protein supplementation on anabolic signaling and rates of myofibrillar and mitochondrial protein synthesis after a single bout of concurrent training. Using a randomized crossover design, eight healthy males were assigned to experimental trials consisting of resistance exercise (8 × 5 leg extension, 80% 1RM) followed by cycling (30 min at approximately 70% V˙O2peak) with either postexercise protein (PRO, 25-g whey protein) or placebo (PLA) ingestion. Muscle biopsies were obtained at rest and at 1 and 4 h after exercise. Akt and mTOR phosphorylation increased 1 h after exercise with PRO (175%-400%, P < 0.01) and was different from PLA (150%-300%, P < 0.001). Muscle RING finger 1 and atrogin-1 messenger RNA (mRNA) were elevated after exercise but were higher with PLA compared with those in PRO at 1 h (50%-315%, P < 0.05), whereas peroxisome proliferator-activated receptor gamma coactivator 1-alpha mRNA increased 4 h after exercise (620%-730%, P < 0.001), with no difference between treatments. Postexercise rates of myofibrillar protein synthesis increased above rest in both trials (75%-145%, P < 0.05) but were higher with PRO (67%, P < 0.05), whereas mitochondrial protein synthesis did not change from baseline. Our results show that a concurrent training session promotes anabolic adaptive responses and increases metabolic/oxidative mRNA expression in the skeletal muscle. PRO ingestion after combined resistance and endurance exercise enhances myofibrillar protein synthesis and attenuates markers of muscle catabolism and thus is likely an important nutritional strategy to enhance adaptation responses with concurrent training.

  18. Sequence determinants of protein aggregation: tools to increase protein solubility

    PubMed Central

    Ventura, Salvador

    2005-01-01

    Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, very often the target protein accumulates into insoluble aggregates in a misfolded and biologically inactive form. Bacterial inclusion bodies are major bottlenecks in protein production and are hampering the development of top priority research areas such structural genomics. Inclusion body formation was formerly considered to occur via non-specific association of hydrophobic surfaces in folding intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria resembles to the well-studied process of amyloid fibril formation. Both processes appear to rely on the formation of specific, sequence-dependent, intermolecular interactions driving the formation of structured protein aggregates. This similarity in the mechanisms of aggregation will probably allow applying anti-aggregational strategies already tested in the amyloid context to the less explored area of protein aggregation inside bacteria. Specifically, new sequence-based approaches appear as promising tools to tune protein aggregation in biotechnological processes. PMID:15847694

  19. Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

    PubMed Central

    Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

    2014-01-01

    The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

  20. Protein Acetylation in Procaryotes Increases Stress Resistance

    PubMed Central

    Ma, Qun; Wood, Thomas K.

    2011-01-01

    Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. PMID:21703240

  1. Protein acetylation in prokaryotes increases stress resistance.

    PubMed

    Ma, Qun; Wood, Thomas K

    2011-07-15

    Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Intra- and extracellular plasminogen activator inhibitor-1 regulate effect of vitronectin against radiation-induced endothelial cell death.

    PubMed

    Hazawa, Masaharu; Yasuda, Takeshi; Saotome-Nakamura, Ai; Tomiyama, Kenichi; Obara, Chizuka; Goto, Takaya; Tajima, Katsushi

    2016-12-01

    Plasminogen activator inhibitor-1 (PAI-1) is induced by radiation resulting in endothelial cell impairment, potentially leading to multiple organ failure. Vitronectin (VN) is a 75-kDa glycoprotein (VN75) cleaved into two forms (VN75 or VN65/10) by furin, which is regulated by intracellular PAI-1. VN protects against radiation-induced endothelial cell death, but the mechanisms involved in VN processing and its interactions with intra- and extracellular PAI-1 remain unclear. We examined these processes in cells in vitro using recombinant proteins or overexpression of VN and PAI-1 genes, including furin-susceptible (T(381)) and furin-resistant VN (A(381)). VN processing was analyzed using a mutant PAI-1 with relatively weaker binding to VN. VN function was evaluated by survival of radiation-damaged endothelial cells. Wild-type, but not mutant PAI-1 inhibited furin-dependent VN processing. Gene transfer revealed that furin-susceptible VN was processed more than the furin-resistant form, but processing of both was inhibited by PAI-1 overexpression. Intracellular PAI-1 formed a complex with VN75 (T(381)) in cells and media, and the VN75 form was secreted preferentially. Only VN75 protected against radiation-induced endothelial cell death, in which its effect was abolished by wild-type but not mutant PAI-1. These findings indicate that intracellular PAI-1 inhibits VN processing and protects against radiation-induced endothelial cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Accessible Surfaces of Beta Proteins Increase with Increasing Protein Molecular Mass More Rapidly than Those of Other Proteins

    PubMed Central

    Glyakina, Anna V.; Bogatyreva, Natalya S.; Galzitskaya, Oxana V.

    2011-01-01

    Here we present a systematic analysis of accessible surface areas and hydrogen bonds of 2554 globular proteins from four structural classes (all-α, all-β, α/β and α+β proteins) that is aimed to learn in which structural class the accessible surface area increases with increasing protein molecular mass more rapidly than in other classes, and what structural peculiarities are responsible for this effect. The beta structural class of proteins was found to be the leader, with the following possible explanations of this fact. First, in beta structural proteins, the fraction of residues not included in the regular secondary structure is the largest, and second, the accessible surface area of packaged elements of the beta-structure increases more rapidly with increasing molecular mass in comparison with the alpha-structure. Moreover, in the beta structure, the probability of formation of backbone hydrogen bonds is higher than that in the alpha helix for all residues of α+β proteins (the average probability is 0.73±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without proline) and α/β proteins, except for asparagine, aspartic acid, glycine, threonine, and serine (0.70±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without the proline residue). There is a linear relationship between the number of hydrogen bonds and the number of amino acid residues in the protein (). PMID:22145047

  4. The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells

    PubMed Central

    1991-01-01

    Constitutive production of cytokines was observed in 3 of 12 gamma/delta T cell lines derived from murine epidermis and correlated with the expression of the C gamma 4, V delta 6 T cell receptor (TCR). After adaptation of one of the lines (T195/BW) to serum-free culture conditions, cessation of the "spontaneous" production of interleukin 4 (IL-4) was observed and IL-4 production could then by induced by the addition of RGD-containing extracellular matrix (ECM) proteins to the culture. The response to the ECM proteins could be completely inhibited by a mAb to the murine vitronectin receptor (VNR). However, the induction of IL-4 production could also be inhibited by anti-CD3 and by an anti-clonotypic mAb to the TCR-gamma/delta of T195/BW. As TCR- gamma/delta loss mutants of T195/BW also failed to respond to ECM proteins, these data demonstrate that engagement of the VNR by its ligand is necessary, but not sufficient, for the induction of IL-4 production. Furthermore, the VNR is expressed by many other T cell clones (both gamma/delta and alpha/beta), none of which produce lymphokines constitutively. Taken together, these observations strongly favor the view that not only is coexpression of the VNR and TCR required for the induction of IL-4 production, but that the TCR must also be engaged by its ligand, most likely a cell surface antigen expressed by the hybridoma itself. PMID:1702138

  5. A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation.

    PubMed

    Min, Seung-Ki; Kang, Hyun Ki; Jung, Sung Youn; Jang, Da Hyun; Min, Byung-Moo

    2017-09-22

    Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with β1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvβ3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.Cell Death and Differentiation advance online publication, 22 September 2017; doi:10.1038/cdd.2017.153.

  6. The 82-plex plasma protein signature that predicts increasing inflammation

    PubMed Central

    Tepel, Martin; Beck, Hans C.; Tan, Qihua; Borst, Christoffer; Rasmussen, Lars M.

    2015-01-01

    The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney transplant recipients and quantified 359 plasma proteins simultaneously using nano-Liquid-Chromatography-Tandem Mass-Spectrometry in individual samples and plasma C-reactive protein on the index day and the next day. Next-day C-reactive protein increased in 59 patients whereas it decreased in 32 patients. The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P < 0.0001). Multivariable logistic regression showed that 82-plex protein signature (P < 0.001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein signature increased net reclassification index with a clinical meaningful 10% increase of risk mainly by the improvement of reclassification of subjects in the event group. An 82-plex plasma protein signature predicts an increase of the inflammatory marker C-reactive protein. PMID:26445912

  7. Identification of vitronectin as an extrinsic inducer of cancer stem cell differentiation and tumor formation.

    PubMed

    Hurt, Elaine M; Chan, King; Serrat, Maria Ana Duhagon; Thomas, Suneetha B; Veenstra, Timothy D; Farrar, William L

    2010-03-31

    There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.

  8. Discovery of new small molecules targeting the vitronectin-binding site of the urokinase receptor that block cancer cell invasion.

    PubMed

    Rea, Vincenza Elena Anna; Lavecchia, Antonio; Di Giovanni, Carmen; Rossi, Francesca Wanda; Gorrasi, Anna; Pesapane, Ada; de Paulis, Amato; Ragno, Pia; Montuori, Nunzia

    2013-08-01

    Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC50 values of 3.6 and 1.2 μmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

  9. Charged ultrafiltration membranes increase the selectivity of whey protein separations.

    PubMed

    Bhushan, S; Etzel, M R

    2009-04-01

    Ultrafiltration is widely used to concentrate proteins, but fractionation of one protein from another is much less common. This study examined the use of positively charged membranes to increase the selectivity of ultrafiltration and allow the fractionation of proteins from cheese whey. By adding a positive charge to ultrafiltration membranes, and adjusting the solution pH, it was possible to permeate proteins having little or no charge, such as glycomacropeptide, and retain proteins having a positive charge. Placing a charge on the membrane increased the selectivity by over 600% compared to using an uncharged membrane. The data were fit using the stagnant film model that relates the observed sieving coefficient to membrane parameters such as the flux, mass transfer coefficient, and membrane Peclet number. The model was a useful tool for data analysis and for the scale up of membrane separations for whey protein fractionation.

  10. Identification of proteins with increased levels in ameloblastic carcinoma.

    PubMed

    García-Muñoz, Alejandro; Bologna-Molina, Ronell; Aldape-Barrios, Beatriz; Licéaga-Escalera, Carlos; Montoya-Pérez, Luis A; Rodríguez, Mario A

    2014-06-01

    The comparative proteomic approach by a combination of 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MS) analysis is an attractive strategy for the discovery of cancer biomarkers and therapeutic targets. The identification of protein biomarkers associated with ameloblastic carcinoma (AC), a malignant epithelial odontogenic tumor, will potentially improve the diagnostic and prognostic accuracy for this malignant neoplasm. The aim of the present study was to identify highly expressed proteins in AC that could be considered as potential biomarkers. The protein profile of an AC was compared with the protein profiles of 3 cases of benign ameloblastoma. Proteins that showed increased levels in AC were identified using MS, and the augmented amount of some of these proteins in the malignant lesion was confirmed by Western blot or immunohistochemistry. We detected a total of 782 spots in the protein profile of AC, and 19 of them, showing elevated levels compared with benign ameloblastoma, were identified using MS. These proteins have been implicated in several cellular functions, such as cell structure, metabolism, stress response, and signal transduction. The increased expression of the identified proteins and the minor expression of some proteins that might inhibit tumor progression could be involved in the evolution from a benign lesion to carcinoma. Copyright © 2014 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  11. Elevated Protein Level Increases Blacks' Risk of Kidney Disease

    MedlinePlus

    ... Español You Are Here: Home → Latest Health News → Article URL of this page: https://medlineplus.gov/news/fullstory_166903.html Elevated Protein Level Increases Blacks' Risk of Kidney Disease Scientists find enhanced protein level is required to trigger ...

  12. INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

    PubMed Central

    Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

  13. Efficiency of the immunome protein interaction network increases during evolution.

    PubMed

    Ortutay, Csaba; Vihinen, Mauno

    2008-04-22

    Details of the mechanisms and selection pressures that shape the emergence and development of complex biological systems, such as the human immune system, are poorly understood. A recent definition of a reference set of proteins essential for the human immunome, combined with information about protein interaction networks for these proteins, facilitates evolutionary study of this biological machinery. Here, we present a detailed study of the development of the immunome protein interaction network during eight evolutionary steps from Bilateria ancestors to human. New nodes show preferential attachment to high degree proteins. The efficiency of the immunome protein interaction network increases during the evolutionary steps, whereas the vulnerability of the network decreases. Our results shed light on selective forces acting on the emergence of biological networks. It is likely that the high efficiency and low vulnerability are intrinsic properties of many biological networks, which arise from the effects of evolutionary processes yet to be uncovered.

  14. Endotoxin increases pulmonary vascular protein permeability in the dog

    SciTech Connect

    Welsh, C.H.; Dauber, I.M.; Weil, J.V.

    1986-10-01

    Endotoxin increases pulmonary vascular permeability consistently in some species but fails to reliably cause injury in the dog. We wondered whether this phenomenon depended on the method of injury assessment, as others have relied on edema measurement; we quantified injury by monitoring the rate of extravascular protein accumulation. /sup 113m/In-labeled protein and /sup 99m/Tc-labeled erythrocytes were injected into anesthetized dogs and monitored by an externally placed lung probe. A protein leak index, the rate of extravascular protein accumulation, was derived from the rate of increase in lung protein counts corrected for changes in intravascular protein activity. After administration of Salmonella enteriditis endotoxin (4 micrograms/kg), the protein leak index was elevated 2.5-fold (41.1 +/- 4.6 X 10(-4) min-1) compared with control (16.0 +/- 2.8 X 10(-4) min-1). In contrast, wet-to-dry weight ratios failed to increase after endotoxin (4.6 +/- 0.8 vs. control values of 4.2 +/- 0.5 g/g dry bloodless lung). However, we observed that endotoxin increased lung dry weight (per unit body weight), which may have attenuated the change in wet-to-dry weight ratios. To determine whether low microvascular pressures following endotoxin attenuated edema formation, we increased pulmonary arterial wedge pressures in five dogs by saline infusion, which caused an increase in wet-to-dry weight ratios following endotoxin but no change in the five controls. We conclude that low dose endotoxin causes pulmonary vascular protein leak in the dog while edema formation is minimal or absent.

  15. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  16. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  17. Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility.

    PubMed

    Kramer, Ryan M; Shende, Varad R; Motl, Nicole; Pace, C Nick; Scholtz, J Martin

    2012-04-18

    Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

    PubMed Central

    Kramer, Ryan M.; Shende, Varad R.; Motl, Nicole; Pace, C. Nick; Scholtz, J. Martin

    2012-01-01

    Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. PMID:22768947

  19. Vitronectin: a promising breast cancer serum biomarker for early diagnosis of breast cancer in patients.

    PubMed

    Hao, Wende; Zhang, Xuhui; Xiu, Bingshui; Yang, Xiqin; Hu, Shuofeng; Liu, Zhiqiang; Duan, Cuimi; Jin, Shujuan; Ying, Xiaomin; Zhao, Yanfeng; Han, Xiaowei; Hao, Xiaopeng; Fan, Yawen; Johnson, Heather; Meng, Di; Persson, Jenny L; Zhang, Heqiu; Feng, XiaoYan; Huang, Yan

    2016-07-01

    Breast cancer is the most common cancer in women worldwide, identification of new biomarkers for early diagnosis and detection will improve the clinical outcome of breast cancer patients. In the present study, we determined serum levels of vitronectin (VN) in 93 breast cancer patients, 30 benign breast lesions, 9 precancerous lesions, and 30 healthy individuals by enzyme-linked immunosorbent assays. Serum VN level was significantly higher in patients with stage 0-I primary breast cancer than in healthy individuals, patients with benign breast lesion or precancerous lesions, as well as those with breast cancer of higher stages. Serum VN level was significantly and negatively correlated with tumor size, lymph node status, and clinical stage (p < 0.05 in all cases). In addition, VN displayed higher area under curve (AUC) value (0.73, 95 % confidence interval (CI) [0.62-0.84]) than carcinoembryonic antigen (CEA) (0.64, 95 % CI [0.52-0.77]) and cancer antigen 15-3 (CA 15-3) (0.69, 95 % CI [0.58-0.81]) when used to distinguish stage 0-I cancer and normal control. Importantly, the combined use of three biomarkers yielded an improvement in receiver operating characteristic curve with an AUC of 0.83, 95 % CI [0.74-0.92]. Taken together, our current study showed for the first time that serum VN is a promising biomarker for early diagnosis of breast cancer when combined with CEA and CA15-3.

  20. The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins.

    PubMed

    Ferraris, Gian Maria Sarra; Schulte, Carsten; Buttiglione, Valentina; De Lorenzi, Valentina; Piontini, Andrea; Galluzzi, Massimiliano; Podestà, Alessandro; Madsen, Chris D; Sidenius, Nicolai

    2014-11-03

    The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch. © 2014 The Authors.

  1. The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins

    PubMed Central

    Ferraris, Gian Maria Sarra; Schulte, Carsten; Buttiglione, Valentina; De Lorenzi, Valentina; Piontini, Andrea; Galluzzi, Massimiliano; Podestà, Alessandro; Madsen, Chris D; Sidenius, Nicolai

    2014-01-01

    The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch. PMID:25168639

  2. Shortening a loop can increase protein native state entropy.

    PubMed

    Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov

    2015-12-01

    Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.

  3. Increased protein intake on controlled oxalate diets does not increase urinary oxalate excretion

    PubMed Central

    Easter, Linda H.; Neiberg, Rebecca; Assimos, Dean G.; Holmes, Ross P.

    2009-01-01

    High animal protein intake is a risk factor for calcium oxalate stone disease. The effect of dietary protein on the urinary excretion of calcium, acid and citrate is well established. However, its effect on oxalate excretion is unclear, due in part to an inadequate control of dietary oxalate intake in previous studies. This relationship warrants clarification due to the proposed important role of the metabolism of amino acids in endogenous oxalate synthesis. In this study, 11 normal subjects consumed controlled oxalate diets containing 0.6, 1.2 and 1.8 g protein/kg body weight/day. The analysis of 24 h urine collections confirmed that as protein intake increased, urinary calcium and glycolate increased and urinary pH and citrate decreased. The increased glycolate excretion was due in part to an increased hydroxyproline, but not glycolate consumption. Total daily urinary oxalate excretion did not change. When indexed to creatinine there was a small but significant decrease in oxalate excretion. This is most likely due to hyperfiltration. These results indicate that as dietary protein intake increases, the catabolism of diet-derived amino acids is not associated with an increased endogenous oxalate synthesis in normal subjects. PMID:19183980

  4. Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation

    PubMed Central

    Bowley, Sheryl R.; Fang, Chao; Merrill-Skoloff, Glenn; Furie, Barbara C.; Furie, Bruce

    2017-01-01

    Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for thrombus formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for thrombus formation. PMID:28218242

  5. Poliovirus protein 2BC increases cytosolic free calcium concentrations.

    PubMed Central

    Aldabe, R; Irurzun, A; Carrasco, L

    1997-01-01

    Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis. PMID:9223520

  6. Wisconsin - Increased corn silage protein with intercropped lablab bean

    USDA-ARS?s Scientific Manuscript database

    Protein supplements for livestock are costly. In recent research in southern WI, lablab bean grown with corn increased forage CP concentration over monoculture corn without compromising forage yield or potential milk production per acre. Corn was intercropped with each of three climbing beans: lab...

  7. Bovine plasma proteins increase virulence of Haemophilus somnus in mice.

    PubMed

    Geertsema, Roger S; Kimball, Richard A; Corbeil, Lynette B

    2007-01-01

    The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen.

  8. Adsorption of fibronectin and vitronectin onto Primaria and tissue culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial cells and BHK-21 fibroblasts.

    PubMed

    Steele, J G; Dalton, B A; Johnson, G; Underwood, P A

    1995-09-01

    The two cell culture substrata, tissue culture polystyrene (TCPS) and Primaria, were compared in order to determine whether a nitrogen-containing surface such as Primaria attracts cells by a different mechanism to an oxygen-containing surface (TCPS). The amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium onto Primaria and TCPS were determined. Primaria adsorbed two- to threefold more Fn than TCPS, but adsorbed similar amounts of Vn from medium containing FBS. The Fn and Vn binding sites on Primaria were distinct, as adsorption was non-competitive between these two proteins. The amounts of Fn and Vn that adsorbed onto the two surfaces were compared to the concentration dependence of the cell attachment activities of Fn and Vn. Whereas the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment, Primaria adsorbed an Fn surface density that was supraoptimal for attachment of human vein endothelial cells and BHK-21 fibroblasts. We conclude that Primaria differs from TCPS in that both Fn and Vn mediate initial cell attachment to Primaria when the culture medium contains FBS, whereas cell attachment to TCPS is dependent upon Vn.

  9. Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection.

    PubMed

    T, Prasada Rao; T, Lakshmi Prasanth; R, Parvathy; S, Murugavel; Devi, Karuna; Joshi, Paritosh

    2017-02-02

    Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix interacts with complement C9. This interaction may modulate innate immunity. Details of Vn-C9 interaction are limited. An assessment of Vn-C9 interaction was made employing goat homologous system. Vn binding to C9 was observed in three different assays. Using recombinant fragments, the C9 binding was mapped to the N-terminus of Vn. Site directed mutagenesis was performed to alter the second RGD sequence (RGD-2) of Vn. Change of R to G or D to A in RGD-2 caused significant decrease in Vn binding to C9 whereas change of R to G in the first RGD motif (RGD-1) had no effect on Vn binding to C9. These results imply that the RGD-2 of goat Vn is involved in C9 binding. In competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 in terms of bacterial pathogenesis was also evaluated. Serum dependent inhibition of E. coli growth was significantly reverted when Vn or its N-fragment were included in the assay. The C-fragment, which did not support C9 binding, also partly nullified serum dependent inhibition of bacterial growth probably through other serum component(s).

  10. Specific protein homeostatic functions of small heat-shock proteins increase lifespan.

    PubMed

    Vos, Michel J; Carra, Serena; Kanon, Bart; Bosveld, Floris; Klauke, Karin; Sibon, Ody C M; Kampinga, Harm H

    2016-04-01

    During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.

  11. Ovarian Steroids Increase Spinogenetic Proteins in the Macaque Dorsal Raphe

    PubMed Central

    Rivera, Heidi M.; Bethea, Cynthia L.

    2012-01-01

    Dendritic spines are the basic structural units of neuronal plasticity. Intracellular signaling cascades that promote spinogenesis have centered on RhoGTPases. We found that ovarian steroids increase gene expression of RhoGTPases (RhoA, Cdc42 and Rac) in laser-captured serotonin neurons. We sought to confirm that the increases observed in gene expression translate to the protein level. In addition, a preliminary study was conducted to determine whether an increase in spines occurs via detection of the spine marker protein, PSD-95. Adult ovariectomized (Ovx) monkeys were treated with estradiol (E), progesterone (P) or E+P for 1 month. Sections through the dorsal raphe nucleus were immunostained for RhoA and Cdc42 (n = 3-4/group). The number and positive pixel area of RhoA-positive cells, and the positive pixel area of Cdc42-positive fibers were determined. Upon combining E and E+P treated groups, there was a significant increase in the average and total cell number and positive pixel area of RhoA-positive cells. E, P and E+P treatments individually or combined, also increased the average and total positive pixel area of Cdc42-positive fibers. With remaining sections from 2 animals in each group, we conducted a preliminary examination of the regulation of PSD-95 protein expression. PSD-95, a postsynaptic scaffold protein, was examined with immunogold silver staining (n = 2/group) and the total number of PSD-95-positive puncta was determined with stereology across 4 levels of the dorsal raphe. E, P and E+P treatment significantly increased the total number of PSD-95-positive puncta. Together, these findings indicate that ovarian steroids act to increase gene and protein expression of two pivotal RhoGTPases involved in spinogenesis and preliminarily indicate that an increased number of spines and/or synapses result from this action. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and in turn, increase serotonin neuronal

  12. Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

    PubMed Central

    Le Loir, Yves; Azevedo, Vasco; Oliveira, Sergio C; Freitas, Daniela A; Miyoshi, Anderson; Bermúdez-Humarán, Luis G; Nouaille, Sébastien; Ribeiro, Luciana A; Leclercq, Sophie; Gabriel, Jane E; Guimaraes, Valeria D; Oliveira, Maricê N; Charlier, Cathy; Gautier, Michel; Langella, Philippe

    2005-01-01

    Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. PMID:15631634

  13. Increasing Protein Charge State When Using Laser Electrospray Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Karki, Santosh; Flanigan, Paul M.; Perez, Johnny J.; Archer, Jieutonne J.; Levis, Robert J.

    2015-05-01

    Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol ( m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

  14. Increased protein intake decreases postnatal growth faltering in ELBW babies.

    PubMed

    Cormack, Barbara Elizabeth; Bloomfield, Frank H

    2013-09-01

    To determine whether purposely designed nutritional guidelines for extremely low birthweight (ELBW; birth weight <1000 g) babies result in protein intakes that meet international consensus recommendations, and whether this results in improved growth from birth to discharge. A prospective cohort study of nutritional intakes and growth in ELBW babies. A tertiary neonatal intensive care unit in New Zealand. 100 ELBW babies who survived for the first month of life, 50 before the introduction of the guideline (Lo Pro) and 50 after (Hi Pro). Introduction of a nutritional guideline aimed at increasing protein intakes to meet international consensus recommendations. Weekly protein intakes over the first month of life and growth until discharge. Hi Pro babies had significantly higher protein intakes in the first month of life than Lo Pro babies (mean (SD), 3.8 (0.3) vs 3.3 (0.4) g/kg.day, p<0.0001) and a significantly greater growth velocity (GV) over the first 30 days after regaining birth weight (19.5 (5.0) vs 16.2 (5.4) g/kg.day, p<0.002). Hi Pro babies had a significantly lesser Z-score change between birth and discharge than Lo Pro babies for weight (0.0 (1.2) vs -0.9 (1.1), p=0.001), length (-0.8 (0.8) vs -1.2 (1.1), p=0.02) and head circumference (-0.2 (1.1) vs -1.1 (1.6), p<0.001). Simple, standardised nutritional guidelines can result in recommended protein intakes for ELBW babies being achieved and result in increased GV. Downward crossing of centiles between birth and discharge, common in ELBW babies, is significantly reduced for weight, length and head circumference.

  15. Protein and carbohydrate supplementation increases aerobic and thermoregulatory capacities

    PubMed Central

    Okazaki, Kazunobu; Goto, Masaki; Nose, Hiroshi

    2009-01-01

    The incidence of heat illness and heat stroke is greater in older than younger people. In this context, exercise training regimens to increase heat tolerance in older people may provide protection against heat illness. Acute increases in plasma volume (PV) improve thermoregulation during exercise in young subjects, but there is some evidence that changes in PV in response to acute exercise are blunted in older humans. We recently demonstrated that protein–carbohydrate (Pro-CHO) supplementation immediately after a bout of exercise increased PV and plasma albumin content (Albcont) after 23 h in both young and older subjects. We also examined whether Pro-CHO supplementation during aerobic training enhanced thermoregulation by increasing PV and Albcont in older subjects. Older men aged ∼68 years exercised at moderate intensity, 60 min day−1, 3 days week−1, for 8 weeks, at ∼19°C, and took either placebo (CNT; 0.5 kcal, 0 g protein kg−1) or Pro-CHO supplement (Pro-CHO; 3.2 kcal, 0.18 g protein kg−1) immediately after exercise. After training, we found during exercise at 30°C that increases in oesophageal temperature (Tes) were attenuated more in Pro-CHO than CNT and associated with enhanced cutaneous vasodilatation and sweating. We also confirmed similar results in young subjects after 5 days of training. These results demonstrate that post-exercise protein and CHO consumption enhance thermoregulatory adaptations especially in older subjects and provide insight into potential strategies to improve cardiovascular and thermoregulatory adaptations to exercise in both older and younger subjects. PMID:19752117

  16. Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions

    PubMed Central

    Chen, Fangmin; Krasity, Benjamin C.; Peyer, Suzanne M.; Koehler, Sabrina; Ruby, Edward G.

    2017-01-01

    ABSTRACT We characterized bactericidal permeability-increasing proteins (BPIs) of the squid Euprymna scolopes, EsBPI2 and EsBPI4. They have molecular characteristics typical of other animal BPIs, are closely related to one another, and nest phylogenetically among invertebrate BPIs. Purified EsBPIs had antimicrobial activity against the squid’s symbiont, Vibrio fischeri, which colonizes light organ crypt epithelia. Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies. Pretreatment under acidic conditions similar to those during symbiosis initiation rendered V. fischeri more resistant to the antimicrobial activity of the proteins. Immunocytochemistry localized EsBPIs to the symbiotic organ and other epithelial surfaces interacting with ambient seawater. The proteins differed in intracellular distribution. Further, whereas EsBPI4 was restricted to epithelia, EsBPI2 also occurred in blood and in a transient juvenile organ that mediates hatching. The data provide evidence that these BPIs play different defensive roles early in the life of E. scolopes, modulating interactions with the symbiont. PMID:28377525

  17. Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences

    PubMed Central

    Green, Lisa; Houck-Loomis, Brian; Yueh, Andrew

    2012-01-01

    Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release. PMID:22718819

  18. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

    PubMed Central

    Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

    2015-01-01

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

  19. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

    PubMed

    Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h(-1) in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

  20. Lotus-leaf-like topography predominates over adsorbed ECM proteins in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) surface/cell interactions.

    PubMed

    Zheng, Jun; Li, Dan; Yuan, Lin; Liu, Xiaoli; Chen, Hong

    2013-06-26

    It is well-known that extracellular matrix (ECM) proteins mediate cell/surface interactions. However, introduction of a specific surface topography may disturb the correlation between ECM proteins adsorption and cells adhesion on a given surface. In present study, lotus-leaf-like topography was introduced on the surface of a biodegradable material, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Protein adsorption and cell interactions with this lotus-leaf-like surface (designated PHBHHx-L) were investigated. Water contact angle data indicated that the hydrophobicity of PHBHHx was enhanced by the introduction of lotus-leaf-like topography. The adsorption of extracellular matrix proteins (fibronectin and vitronectin) on PHBHHx-L was measured by enzyme linked immunosorbent assay (ELISA). Compared with flat PHBHHx, adsorption on the PHBHHx-L surface increased by ~260% for fibronectin and ~40% for vitronectin. In contrast, fibroblast and endothelial cell adhesion and proliferation were reduced on the PHBHHx-L compared to the flat polymer surface. These results suggest that the inhibition of cell adhesion and proliferation caused by the lotus-leaf-like topography dominates over the effect of the adsorbed adhesive proteins in promoting adhesion and proliferation. It can be concluded that the lotus-leaf-like topography plays a dominant role in cell/PHBHHx-L interactions. The present findings indicate the complexity of the interplay among surface topography, adsorbed proteins, and cell-surface interactions.

  1. Increased protein glycation in fructosamine 3-kinase-deficient mice

    PubMed Central

    da-Cunha, Maria VEIGA; Jacquemin, Patrick; Delpierre, Ghislain; Godfraind, Catherine; Théate, Ivan; Vertommen, Didier; Clotman, Frédéric; Lemaigre, Frédéric; Devuyst, Olivier; Van Schaftingen, Emile

    2006-01-01

    Amines, including those present on proteins, spontaneously react with glucose to form fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-ϵ-lysines and which is potentially involved in protein repair. Fn3k−/− mice looked healthy and had normal blood glucose and serum fructosamine levels. However, their level of haemoglobin-bound fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/− mice. Other intracellular proteins were also significantly more glycated in Fn3k−/− mice in erythrocytes (1.8–2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2–1.8-fold), indicating that FN3K removes fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-ϵ-lysine was 10–20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k−/− mice, indicating that food is the main source of urinary fructose-ϵ-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-ϵ-lysine. However, in starved animals, the urinary excretion of fructose-ϵ-lysine was 2.5-fold higher in Fn3k−/− mice compared with Fn3k+/+ or Fn3k+/− mice. Furthermore, a marked increase (5–13-fold) was observed in the concentration of free fructose-ϵ-lysine in tissues of fed Fn3k−/− mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-ϵ-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-ϵ-lysine. PMID:16819943

  2. Compound Pollen Protein Nutrient Increases Serum Albumin in Cirrhotic Rats

    PubMed Central

    Shi, Hong Bo; Kong, Ming; Chen, Gong; Zhao, Jun; Shi, Hong Lin; Chen, Yu; Rowan, Frank G

    2010-01-01

    Background Malnutrition, especially protein-calorie malnutrition, is common in patients with liver cirrhosis. When in the status of malnutrition, the complications increase, liver function deteriorates, and the prognosis of patients with liver cirrhosis worsens. Hence, nutritional support and treatment is essential in patients with liver cirrhosis. Previous studies suggested that compound nutrition based on pollen can improve liver function, and can be a basic nutrient for patients with liver cirrhosis. However, the nutritional support based on pollen for malnutrition of cirrhotic patients needs to be further evaluated. In this study, we investigated the nutritional support of Noveliver, a new compound pollen protein nutrient, in the cirrhotic rats induced by carbon tetrachloride (CCl4). Methods The cirrhotic rats induced by CCl4 were treated with Noveliver in different doses, and treated with a regular compound pollen nutrient, untreated cirrhotic rats and normal rats were used as controls. Serum albumin were measured before and after the nutritional treatment in each group. At the same time, liver function, cytokines and pathological changes were also determined. Results In the second week of nutritional treatment, the levels of serum albumin in normal control group, low dose noveliver group, high dose noveliver group, compound protein pollen group and spontaneous recovery group were 35.67 ± 1.42, 33.07 ± 1.27, 32.27 ± 1.50, 30.53 ± 0.25, 24.53 ± 3.56 (g/L), respectively, the differences among the groups were significant (F = 14.007, P = 0.000); The levels of serum albumin in low dose Noveliver group, high dose Noveliver group and the compound protein pollen group were higher than that in the spontaneous recovery group (P = 0.000, 0.001, 0.003, respectively). In the second week of nutritional treatment, the serum levels of HGF in normal control group, low dose Noveliver group, high dose Noveliver group, compound protein pollen group and spontaneous recovery

  3. Bactericidal/permeability increasing protein: a multifaceted protein with functions beyond LPS neutralization.

    PubMed

    Balakrishnan, Arjun; Marathe, Sandhya A; Joglekar, Madhura; Chakravortty, Dipshikha

    2013-01-01

    Bactericidal permeability increasing protein (BPI), a 55-60 kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

  4. Ethanol increases affinity of protein kinase C for phosphatidylserine

    SciTech Connect

    Chin, J.H.

    1986-03-01

    Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition of calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.

  5. Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin.

    PubMed

    Kobayashi, Masaki; Kawabata, Keigo; Kusaka-Kikushima, Ayumi; Sugiyama, Yoshinori; Mabuchi, Tomotaka; Takekoshi, Susumu; Miyasaka, Muneo; Ozawa, Akira; Sakai, Shingo

    2016-06-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-β antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-β-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process.

  6. New reagents for increasing ESI multiple charging of proteins and protein complexes.

    PubMed

    Lomeli, Shirley H; Peng, Ivory X; Yin, Sheng; Loo, Rachel R Ogorzalek; Loo, Joseph A

    2010-01-01

    The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.

  7. Pentraxin 3 increase is much less pronounced than C-reactive protein increase after surgical procedures.

    PubMed

    Åkerfeldt, Torbjörn; Larsson, Anders

    2011-10-01

    Pentraxin 3 is an acute phase marker that belongs to the same protein family as C-reactive protein (CRP). The aim of this study was to compare the acute phase reactions of pentraxin 3 and CRP in humans. High sensitivity CRP and pentraxin 3 were analyzed in blood samples from orthopedic surgery (n = 29) and coronary bypass patients (n = 21). The samples were collected prior to surgery and 4 and 30 days after surgery, respectively. Both CRP and pentraxin 3 were significantly increased at day 4. Median pentraxin 3 values increased from 4,021 to 7,459 pg/mL in the orthopedic group and from 4,637 pg/mL to 10,419 pg/mL in the coronary bypass group while CRP increased from 6.3 mg/L to 151.6 mg/L and from 5.7 mg/L to 176.3 mg/L in the same groups. Pentraxin 3 shows a much smaller increment in humans in comparison with CRP.

  8. A Low Protein Diet Increases the Hypoxic Tolerance in Drosophila

    PubMed Central

    Vigne, Paul; Frelin, Christian

    2006-01-01

    Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O2) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses. PMID:17183686

  9. A low protein diet increases the hypoxic tolerance in Drosophila.

    PubMed

    Vigne, Paul; Frelin, Christian

    2006-12-20

    Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O(2)) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses.

  10. Increased heat shock protein expression after stress in Japanese quail.

    PubMed

    Hoekstra, K A; Iwama, G K; Nichols, C R; Godin, D V; Cheng, K M

    1998-12-01

    Heat shock proteins (HSPs) have been shown to provide information on the biological impact of environmental stress to organisms, yet none have investigated the HSP response to stress in birds. Japanese quail were exposed to seven different stressors (mild restraint, loud noise, inescapable irritation, cold temperature, isolation in darkness, and two stressful social situations) and expression of HSP30, 60, 70, and 90 in heart, liver, lung, kidney and gonads was examined. Tonic Immobility (TI) tests were also conducted to assess whether the stressors increased fear response. Increased expression of HSP70 was found in the myocardial tissue of birds exposed to loud noise, inescapable irritation, cold temperature, and isolation in darkness. Increased expression of other HSPs was not apparent in the heart or any of the other all tissues examined. Longer TI was observed only in birds exposed to the noise stress. Evidence is presented that a fairly wide range of stressors caused increased expression of HSP70 in the Japanese quail myocardial tissue and that HSPs may provide useful biomarkers for the study of environmental stress in birds.

  11. Erythropoietin administration increases splenic erythroferrone protein content and liver TMPRSS6 protein content in rats.

    PubMed

    Gurieva, Iuliia; Frýdlová, Jana; Rychtarčíková, Zuzana; Vokurka, Martin; Truksa, Jaroslav; Krijt, Jan

    2017-02-28

    Erythroferrone (ERFE) and TMPRSS6 are important proteins in the regulation of iron metabolism. The objective of the study was to examine splenic ERFE and liver TMPRSS6 synthesis in rats treated with a combination of iron and erythropoietin (EPO). EPO was administered to female Wistar rats at 600U/day for four days, iron-pretreated rats received 150mg of iron before EPO treatment. Content of ERFE and TMPRSS6 proteins was determined by commercial antibodies. Iron pretreatment prevented the EPO-induced decrease in hepcidin expression. Content of phosphorylated SMAD 1,5,8 proteins was decreased in the liver by both EPO and iron plus EPO treatment. Fam132b expression in the spleen was increased both by EPO and iron plus EPO treatments; these treatments also significantly induced splenic Fam132a expression. ERFE protein content in the spleen was increased both by EPO and iron plus EPO to a similar extent. EPO administration increased TMPRSS6 content in the plasma membrane-enriched fraction of liver homogenate; in iron-pretreated rats, this increase was abolished. The results confirm that iron pretreatment prevents the EPO-induced decrease in liver Hamp expression. This effect probably occurs despite high circulating ERFE levels, since EPO-induced ERFE protein synthesis is not influenced by iron pretreatment.

  12. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    PubMed

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-04-16

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well.

  13. Increasing dietary crude protein does not increase the methionine requirement in kittens.

    PubMed

    Strieker, M J; Morris, J G; Kass, P H; Rogers, Q R

    2007-12-01

    The objective of this study was to determine if the methionine (met) requirement of kittens is correlated with the concentration of dietary crude protein (CP). The study used 48 male kittens in two replications of six 4 x 4 Latin squares, each representing one concentration of met (1.5, 2.5, 3.5, 4.5, 6.0 or 9.0 g/kg diet) with four CP concentrations (150, 200, 300 and 500 g/kg diet) in 2-week periods. Cystine was present in the lowest CP diet at 5.3 g/kg diet and increased as dietary CP increased. Body weight gain, food intake, nitrogen balance and plasma amino acids, glucose, insulin, cortisol, somatomedin C, T(3) and T(4) concentrations on day 12 were measured. From breakpoint analysis of the nitrogen retention curves, the met requirement of kittens was found to be 3.1, 3.8, 3.1 and 2.4 g met/kg for the 150, 200, 300 and 500 g CP/kg diets, respectively. When met was limiting (1.5 or 2.5 g/kg diet), increasing dietary CP did not decrease, but rather increased food intake, body weight gain and nitrogen retention. Plasma met concentrations increased as dietary met increased and at 2.5-3.5 g met/kg diet were not different among kittens fed the various CP diets. Total plasma T(3) and T(4) increased significantly as dietary CP increased in kittens given the 2.5 and 4.5 g met/kg diets. Results indicate that food intake and possibly altered hormonal secretion play a role in this growth response. In conclusion, the met requirement of growing kittens, unlike omnivores and herbivores studied, was not positively correlated with the concentration of dietary CP.

  14. Protein Structure, Function Set for Explosive Increase in Understanding.

    ERIC Educational Resources Information Center

    Chemical and Engineering News, 1986

    1986-01-01

    Cites advances in x-ray diffraction, nuclear magnetic resonance, computer modeling, and display to guide the design and analysis of protein structures. Reviews recent advances in knowledge, synthesis techniques, and theory of proteins. (JM)

  15. Protein Structure, Function Set for Explosive Increase in Understanding.

    ERIC Educational Resources Information Center

    Chemical and Engineering News, 1986

    1986-01-01

    Cites advances in x-ray diffraction, nuclear magnetic resonance, computer modeling, and display to guide the design and analysis of protein structures. Reviews recent advances in knowledge, synthesis techniques, and theory of proteins. (JM)

  16. Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3

    PubMed Central

    1994-01-01

    A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but

  17. One-step seeding of neural stem cells with vitronectin-supplemented medium for high throughput screening assays

    PubMed Central

    Dai, Sheng; Li, Rong; Long, Yan; Titus, Steve; Zhao, Jinghua; Huang, Ruili; Xia, Menghang; Zheng, Wei

    2017-01-01

    Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research. PMID:27647668

  18. Both protein adsorption and aggregation contribute to shear yielding and viscosity increase in protein solutions.

    PubMed

    Castellanos, Maria Monica; Pathak, Jai A; Colby, Ralph H

    2014-01-07

    A combination of sensitive rotational rheometry and surface rheometry with a double-wall ring were used to identify the origins of the viscosity increase at low shear rates in protein solutions. The rheology of two high molecular weight proteins is discussed: Bovine Serum Albumin (BSA) in a Phosphate Buffered Saline solution and an IgG1 monoclonal antibody (mAb) in a formulation buffer containing small quantities of a non-ionic surfactant. For surfactant-free BSA solutions, the interfacial viscosity dominates the low shear viscosity measured in rotational rheometers, while the surfactant-laden mAb solution has an interfacial viscosity that is small compared to that from aggregation in the bulk. A viscoelastic film forms at the air/water interface in the absence of surfactant, contributing to an apparent yield stress (thus a low shear viscosity increase) in conventional bulk rheology measurements. Addition of surfactant eliminates the interfacial yield stress. Evidence of a bulk yield stress arising from protein aggregation is presented, and correlated with results from standard characterization techniques used in the bio-pharmaceutical industry. The protein film at the air/water interface and bulk aggregates both lead to an apparent viscosity increase and their contributions are quantified using a dimensionless ratio of the interfacial and total yield stress. While steady shear viscosities at shear rates below ∼1 s(-1) contain rich information about the stability of protein solutions, embodied in the measured yield stress, such low shear rate data are regrettably often not measured and reported in the literature.

  19. Protein Primary Structure of the Vaccinia Virion at Increased Resolution

    PubMed

    Ngo, Tuan; Mirzakhanyan, Yeva; Moussatche, Nissin; Gershon, Paul David

    2016-11-01

    Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins.

  20. A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction

    PubMed Central

    Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

    2015-01-01

    The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the

  1. A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

    PubMed

    Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O

    2015-06-19

    The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the

  2. Increasing dietary crude protein does not increase the essential amino acid requirements of kittens.

    PubMed

    Strieker, M J; Morris, J G; Rogers, Q R

    2006-08-01

    Essential amino acid (EAA) requirements of omnivores and herbivores (e.g. chicks, lambs, pigs and rats) are directly related to the concentration of dietary crude protein (CP). When an EAA is limiting in the diet, addition of a mixture of EAA lacking the limiting one (which increases dietary CP) results in a decrease in food intake and weight gain. This interaction has been referred to as an AA imbalance and has not been studied in depth in strict carnivores. The objectives of these experiments were to examine the effects on growing kittens (2-week periods) of the addition to diets of a mixture of AA lacking the limiting one. The control diets were at the requirement of the respective limiting EAA (or about 85% of the 1986 National Research Council requirement). In experiment 1, with the dietary EAAs at the minimally determined requirements, the concentration of the essential or dispensable amino acids was increased to determine if CP or an EAA was limiting. Results of growth rates (n = 12) and plasma AA concentrations indicated that tryptophan was limiting, but increased body weight gain also occurred when the concentration of CP was increased as dispensable amino acids without additional tryptophan. Experiment 1 was repeated in experiment 2 using a crossover design. Again, when tryptophan was limiting additional concentrations of dispensable AAs increased body weight gain. This response is the opposite of that in herbivores and omnivores. Experiment 3 consisted of 10 separate crossover trials, one for each of the 10 EAA and examined the effect of two concentrations of dietary CP (200 and 300 g CP/kg diet) on body weight gain of kittens (n = 8) offered diets limiting in each respective EAA. Body weight gain was numerically greater when diets contained 300 g CP/kg than 200 g CP/kg for eight of 10 EAAs (p < 0.05 for only isoleucine and threonine) when each amino acid was limiting. This response is the reverse of that which occurs in chicks, lambs, pigs and rats when

  3. Protein consumption following aerobic exercise increases whole-body protein turnover in older adults.

    PubMed

    Murphy, Cheryl; Miller, Benjamin F

    2010-10-01

    Research measuring whole-body protein turnover (WBPT) after both exercise and nutrition has generally focused on resistance exercise; however, there is a paucity of data regarding the effect of postaerobic exercise nutrition, especially in older adults. It is not known if postexercise protein feeding has a beneficial effect on protein turnover after low- to moderate-intensity exercise. We investigated whether consuming protein plus carbohydrate (PRO) immediately after an acute bout of aerobic exercise has an additive effect over carbohydrate alone (CHO) on WBPT in older individuals. Twelve healthy older adults (age, 59 ± 4 years) were studied on 2 separate occasions after 1 h of exercise at approximately 50% of maximal rate of oxygen uptake, followed by 4 h of recovery. Immediately following exercise, subjects ingested a CHO (60 g) or an isocaloric PRO beverage (40 g carbohydrate, 20 g whey protein). Whole-body protein metabolism was determined using [1-13C]leucine infusion (60 mg prime; 75 mgh(-1) continuous), and sampling blood and expired breath. Rates of whole-body leucine appearance and oxidation, and nonoxidative leucine disposal during the third and fourth hours of postexercise recovery were higher in the PRO group (2.51 ± 0.55, 0.78 ± 0.37, and 1.71 ± 0.44 micromol kg(-1)·min(-1), respectively) than in the CHO group (1.81 ± 0.27, 0.33 ± 0.14, and 1.47 ± 0.25 micromol kg(-1)·min-1, respectively; p = 0.001). Our results indicate that consumption of a PRO beverage after aerobic exercise increased WBPT to a greater extent than a CHO beverage.

  4. A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity

    PubMed Central

    Xu, Xiaojie; Fan, Zhongyi; Liang, Chaoyang; Li, Ling; Wang, Lili; Liang, Yingchun; Wu, Jun; Chang, Shaohong; Yan, Zhifeng; Lv, Zhaohui; Fu, Jing; Liu, Yang; Jin, Shuai; Wang, Tao; Hong, Tian; Dong, Yishan; Ding, Lihua; Cheng, Long; Liu, Rui; Fu, Shenbo; Jiao, Shunchang; Ye, Qinong

    2017-01-01

    Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity. PMID:28094252

  5. Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein.

    PubMed Central

    Horwitz, A H; Williams, R E; Nowakowski, G

    1995-01-01

    Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium. PMID:7822017

  6. Significant increase of chromosomal damage in protein energy malnutrition.

    PubMed

    el-Ghazali, S; Mikhail, M; Awadallah, M; Shatla, H

    1990-12-01

    The present study attempts to investigate the impact of protein energy malnutrition (PEM) on chromosomes. Twenty infants suffering from marasmus and kwashiorkor were chosen from the in-patient paediatric hospital of Ain Shams University. They were matched by age and sex and socioeconomic status to ten healthy infants (controls). Whole blood lymphocytic cultures were set up for every infant and harvested after 48 h incubation. The frequency of chromosomal aberrations was found to be nearly nine times greater among infants with PEM compared to controls (6.7% versus 0.7% respectively); the difference was statistically highly significant, P less than 0.001. The recorded aberrations were mainly chromatid gaps and isogaps. In addition, chromatid breaks, dicentrics and acentric fragments were observed. Moreover, the younger the age of the infant the greater the frequency of chromosomal aberrations. The results direct our attention to the genetic risk effect of PEM.

  7. The increasing value of plant-made proteins.

    PubMed

    Sack, Markus; Hofbauer, Anna; Fischer, Rainer; Stoger, Eva

    2015-04-01

    The production of high-value proteins in plants is maturing, as shown by the recent approval of innovative products and the latest studies that showcase plant-based production systems using technologies and approaches that are well established in other fields. These include host cell engineering, medium optimization, scalable unit operations for downstream processing (DSP), bioprocess optimization and detailed cost analysis. Product-specific benefits of plant-based systems have also been exploited, including bioencapsulation and the mucosal delivery of minimally processed topical and oral products with a lower entry barrier than pharmaceuticals for injection. Success stories spearheaded by the FDA approval of Elelyso developed by Protalix have revitalized the field and further interest has been fueled by the production of experimental Ebola treatments in plants.

  8. Manipulating corn germplasm to increase recombinant protein accumulation.

    PubMed

    Hood, Elizabeth E; Devaiah, Shivakumar P; Fake, Gina; Egelkrout, Erin; Teoh, Keat Thomas; Requesens, Deborah Vicuna; Hayden, Celine; Hood, Kendall R; Pappu, Kameshwari M; Carroll, Jennifer; Howard, John A

    2012-01-01

    Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.

  9. Sex differences in snack food reinforcement in response to increasing dietary protein

    USDA-ARS?s Scientific Manuscript database

    BRACKGROUND: Protein is posited to play a dynamic role in energy balance and reward-driven eating behavior. However, little is known about the effect of increasing protein intake on snack food reinforcement. OBJECTIVE: We sought to determine the extent to which increasing dietary protein changes th...

  10. Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

    PubMed

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

    2016-09-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P < 0.01). Myofibrillar protein synthesis rates increased after ingesting MCas-35 (P < 0.01) and were higher after ingesting MCas-35 (0.050% ± 0.005%/h) than after WPH-35 (0.032% ± 0.004%/h) (P = 0.03). The postprandial increase in plasma leucine concentrations was greater after ingesting Whey-35 than after WPH-60 (peak value: 580 ± 18 compared with 378 ± 10 μM, respectively; P < 0.01), despite similar leucine contents (4.4 g leucine). Nevertheless, the ingestion of WPH-60 increased myofibrillar protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an

  11. Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

    SciTech Connect

    Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.; Machius, Mischa; Szyperski, Thomas; Kuhlman, Brian

    2015-10-15

    Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).

  12. The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

    PubMed Central

    Rodríguez-Almazán, Claudia; Torner, Francisco J.; Costas, Miguel; Pérez-Montfort, Ruy; de Gómez-Puyou, Marieta Tuena; Puyou, Armando Gómez

    2007-01-01

    The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins. PMID:17551578

  13. Menopause increases the iron storage protein ferritin in skin.

    PubMed

    Pelle, Edward; Jian, Jinlong; Zhang, Qi; Muizzuddin, Neelam; Yang, Qing; Dai, Jisen; Maes, Daniel; Pernodet, Nadine; Yarosh, Daniel B; Frenkel, Krystyna; Huang, Xi

    2013-01-01

    Menstruation and desquamation are important routes for humans to excrete iron. Because menstruation is no longer available in postmenopausal women, in the present study, we examined whether iron accumulates more in postmenopausal skin than in premenopausal skin. Skin biopsy samples were obtained from six pre- and six postmenopausal Caucasian women. Iron levels in the form of ferritin were 42% higher, but vascular endothelial growth factor and total antioxidant capacity were 45% and 34% lower in postmenopausal skin (58.8 ± 1.3 years old) than in premenopausal skin (41.6 ± 1.7 years old), respectively. Moreover, in vitro cultured normal human epidermal keratinocytes had surprisingly high levels of ferritin when compared to immortalized human breast epithelial MCF-10A cells or human liver HepG2 cancer cells. Our results indicate that skin is a cellular repository of iron and that menopause increases iron in skin and, thus, may contribute to the manifestation of accelerated skin aging and photo aging after menopause.

  14. [Increased fibrinolytic activity during cardiopulmonary bypass is caused by activated protein C system].

    PubMed

    Gando, S; Tedo, I; Masio, H; Goda, Y; Kawahigashi, H

    1994-06-01

    To examine the hypothesis that activated protein C system during cardiopulmonary bypass surgery may increase fibrinolytic activity during cardiopulmonary bypass, protein C activity, protein C antigen and thrombomodulin of sixteen patients undergoing elective cardiopulmonary bypass surgery were investigated after induction of anesthesia, before and after cardiopulmonary bypass, and at the end of operation. Protein C activity decreased and thrombomodulin increased significantly after the cardiopulmonary bypass. There were no significant correlations of thrombomodulin with protein C activity and protein C antigen. In conclusion, we have demonstrated that protein C system is activated and circulating thrombomodulin appears in the systemic circulation during cardiopulmonary bypass surgery and this enhanced activation of protein C system is possibly related to the reported increase of fibrinolytic activity during cardiopulmonary bypass.

  15. Exercise increases utrophin protein expression in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Gordon, Bradley S; Lowe, Dawn A; Kostek, Matthew C

    2014-06-01

    Duchenne muscular dystrophy (DMD) is a lethal genetic disease caused by mutations in the dystrophin gene resulting in chronic muscle damage, muscle wasting, and premature death. Utrophin is a dystrophin protein homologue that increases dystrophic muscle function and reduces pathology. Currently, no treatments that increase utrophin protein expression exist. However, exercise increases utrophin mRNA expression in healthy humans. Therefore, the purpose was to determine whether exercise increases utrophin protein expression in dystrophic muscle. Utrophin protein was measured in the quadriceps and soleus muscles of mdx mice after 12 weeks of voluntary wheel running exercise or sedentary controls. Muscle pathology was measured in the quadriceps. Exercise increased utrophin protein expression 334 ± 63% in the quadriceps relative to sedentary controls. Exercise increased central nuclei 4 ± 1% but not other measures of pathology. Exercise may be an intervention that increases utrophin expression in patients with DMD. Copyright © 2013 Wiley Periodicals, Inc.

  16. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    PubMed

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

  17. Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability.

    PubMed

    Prashar, Deepali; Cui, DaWei; Bandyopadhyay, Debjyoti; Luk, Yan-Yeung

    2011-11-01

    This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).

  18. Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

    PubMed Central

    Boston, R S; Fontes, E B; Shank, B B; Wrobel, R L

    1991-01-01

    Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein. PMID:1840924

  19. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  20. Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

    PubMed Central

    Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.; Machius, Mischa; Szyperski, Thomas; Kuhlman, Brian

    2012-01-01

    Summary Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 1-2 Å, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point > 140 °C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 Å). PMID:22632833

  1. Whey protein supplementation increases methionine intake but not homocysteine plasma concentration in rats.

    PubMed

    Deminice, Rafael; Comparotto, Hugo; Jordao, Alceu Afonso

    2015-01-01

    The purpose of this study was to examine the effects of whey protein supplementation on homocysteine (Hcy) metabolism and liver oxidative stress in rats. Twenty-four rats were divided into 3 groups (n = 8) to receive one of the following diets for 4 weeks: control diet (C), whey protein-composed diet (WP), and whey protein-supplemented diet (WPS). The C and WP diets consisted of AIN-93 with 20% casein and 20% whey protein as protein source, respectively. WPS was AIN-93 (20% casein) supplemented by the addition of 20% (w/w) whey protein. Four weeks of ingesting a WPS diet resulted in a significantly higher (P < 0.05) total protein and methionine intakes. Although a significant increase (P < 0.05) in the hepatic S-adenosylmethionine and S-adenosylhomocysteine levels occurred in WPS group compared with C and WP, no significant change was observed in plasma Hcy concentration between groups. Furthermore, the levels of lipid hydroperoxides and advanced oxidation protein products, known liver oxidative stress markers, were increased in the WPS group compared with the C group. In addition, no change in glutathione liver concentration was observed in any of the groups studied. In conclusion, whey protein supplementation increases methionine intake substantially; however, it does not change plasma Hcy concentrations. On the other hand, increased hepatic oxidative stress markers were observed in whey protein supplemented rats were probably due to high protein intake.

  2. Increased nitration and carbonylation of proteins in MRL +/+ mice exposed to trichloroethene: Potential role of protein oxidation in autoimmunity

    SciTech Connect

    Wang Gangduo; Wang Jianling; Ma Huaxian; Khan, M. Firoze

    2009-06-01

    Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4{sup th} day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases ({approx}3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-{gamma}) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

  3. Increased Nitration and Carbonylation of Proteins in MRL +/+ Mice Exposed to Trichloroethene: Potential Role of Protein Oxidation in Autoimmunity

    PubMed Central

    Wang, Gangduo; Wang, Jianling; Ma, Huaxian; Firoze Khan, M.

    2009-01-01

    Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4th day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases (~3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-γ) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs. PMID:19332086

  4. Post-weaning protein malnutrition increases blood pressure and induces endothelial dysfunctions in rats.

    PubMed

    de Belchior, Aucelia C S; Angeli, Jhuli K; Faria, Thaís de O; Siman, Fabiana D M; Silveira, Edna A; Meira, Eduardo F; da Costa, Carlos P; Vassallo, Dalton V; Padilha, Alessandra S

    2012-01-01

    Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10(-10)-3.10(-4) M) was similar in both groups. Endothelium removal or L-NAME (10(-4) M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001-300 µg) and the relaxation to acetylcholine (10(-10)-10(-3) M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model.

  5. Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats

    PubMed Central

    Siman, Fabiana D. M.; Silveira, Edna A.; Meira, Eduardo F.; da Costa, Carlos P.; Vassallo, Dalton V.; Padilha, Alessandra S.

    2012-01-01

    Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model. PMID:22529948

  6. Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity.

    PubMed

    Frizzell, Norma; Thomas, Sonia A; Carson, James A; Baynes, John W

    2012-07-15

    2SC [S-(2-succino)-cysteine] is a chemical modification formed by a Michael addition reaction of fumarate with cysteine residues in proteins. Formation of 2SC, termed 'succination' of proteins, increases in adipocytes grown in high-glucose medium and in adipose tissues of Type 2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30 mM compared with 5 mM) caused a significant increase in cellular ATP/ADP, NADH/NAD+ and Δψm (mitochondrial membrane potential). There was also a significant increase in the cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD+ and subsequent inhibition of tricarboxylic acid cycle NAD+-dependent dehydrogenases. Chemical uncouplers, which dissipated Δψm and reduced the NADH/NAD+ ratio, also decreased the fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD+ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.

  7. Protein malnutrition increases plasma adrenocorticotropin and anterior pituitary proopiomelanocortin messenger ribonucleic acid in the rat.

    PubMed

    Jacobson, L; Zurakowski, D; Majzoub, J A

    1997-03-01

    The mechanism by which protein malnutrition increases circulating glucocorticoids is unclear. To determine whether ACTH synthesis and secretion also increase in protein malnutrition, rats were sham adrenalectomized or adrenalectomized and replaced with varying amounts of corticosterone before dietary protein deprivation. Pair-fed rats served as controls for reduced voluntary food intake in protein-deprived rats. Dietary protein deficiency, but not pair-feeding, increased resting plasma corticosterone in sham-adrenalectomized rats. Restraint-induced ACTH secretion was not inhibited by the increased basal corticosterone levels in protein-deficient rats. When increases in corticosterone were eliminated by adrenalectomy or controlled by adrenalectomy with low level corticosterone replacement, increases in resting plasma ACTH and anterior pituitary POMC messenger RNA expression occurred with protein deprivation that could be statistically discriminated by regression analysis from changes due to caloric restriction (pair-feeding) and overt glucocorticoid feedback resistance. We conclude that protein malnutrition increases pituitary-adrenocortical activity at least in part by specifically increasing the drive for ACTH synthesis and secretion.

  8. Increased in vitro phosphorylation of rat liver nucleolar proteins following triiodothyronine administration.

    PubMed

    Fugassa, E; Gallo, G; Pertica, M

    1976-11-15

    It has been shown that triiodothyronine (Ta) administration to thyroidectomized rats induces an increase in the in vitro net 32P uptake into liver nucleolar proteins. Such an increase depends on a stimulation of the nucleolus-associated protein kinase activity and not on a lower dephosphorylation rate.

  9. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    NASA Technical Reports Server (NTRS)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  10. Threonine utilization for synthesis of acute phase proteins, intestinal proteins, and mucins is increased during sepsis in rats.

    PubMed

    Faure, Magali; Choné, Frédérique; Mettraux, Christine; Godin, Jean-Philippe; Béchereau, Fabienne; Vuichoud, Jacques; Papet, Isabelle; Breuillé, Denis; Obled, Christiane

    2007-07-01

    We hypothesized that the dietary threonine demand for the anabolic response may be increased more than that of other essential amino acids during sepsis. Using a flooding dose of either L-[1 -13C]valine or L-[U -13C]threonine, we measured valine and threonine utilization for syntheses of plasma proteins (minus albumin), and wall, mucosal, and mucin proteins of the small intestine in infected (INF; d 2 and d 6 of postinfection) and control pair-fed (PF) rats. At d 2, the protein absolute synthesis rate (ASR) of INF rats was 21% (mucins) to 41% (intestinal wall) greater than that of PF when measured using valine as tracer, and 45% (mucosa) to 113% (mucins) greater than that of PF when measured with threonine as tracer. Plasma protein ASR was higher in INF than in PF rats, reaching 5- to 6-fold the value of PF. The utilization of both amino acid tracers for the protein synthesis was significantly increased by the infection in all compartments studied. The daily increased absolute threonine utilization for protein synthesis in gut wall plus plasma proteins was 446 micromol/d compared with 365 micromol/d for valine, and it represented 2.6 times the dietary threonine intake of rats at d 2. Most changes in protein ASR and threonine utilization observed at d 6 of postinfection were limited. In conclusion, sepsis increased the utilization of threonine for the anabolic splanchnic response. Because this threonine requirement is likely covered by muscle protein mobilization, increasing the threonine dietary supply would be an effective early nutritional management for patients with sepsis.

  11. Effect of increasing dietary metabolizable protein on nitrogen efficiency in Holstein dairy cows

    PubMed Central

    Imran, Muhammad; Pasha, Talat Naseer; Shahid, Muhammad Qamer; Babar, Imran; Naveed ul Haque, Muhammad

    2017-01-01

    Objective The objective of the study was to determine the effects of increasing levels of metabolizable protein (MP) on lactation performance and nitrogen (N) efficiencies in lactating dairy cows. Methods Nine multiparous cows in mid lactation [113±25 days in milk] received three treatments in a 3×3 Latin square design with a period length of 21 days. The treatments were three diets, designed to provide similar energy and increasing supply of MP (g/d) (2,371 [low], 2,561 [medium], and 2,711 [high] with corresponding crude protein levels [%]) 15.2, 18.4, and 20.9, respectively. Results Increasing MP supplies did not modify dry matter intake, however, it increased milk protein, fat, and lactose yield linearly. Similarly, fat corrected milk increased linearly (9.3%) due to an increase in both milk yield (5.2%) and milk fat content (7.8%). No effects were observed on milk protein and lactose contents across the treatments. Milk nitrogen efficiency (MNE) decreased from 0.26 to 0.20; whereas, the metabolic efficiency of MP decreased from 0.70 to 0.60 in low to high MP supplies, respectively. The concentration of blood urea nitrogen (BUN) increased linearly in response to increasing MP supplies. Conclusion Increasing MP supplies resulted in increased milk protein yield; however, a higher BUN and low MNE indicated an efficient utilization of dietary protein at low MP supplies. PMID:28002937

  12. Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis.

    PubMed

    Duensing, T D; Wing, J S; van Putten, J P

    1999-09-01

    Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.

  13. STRING v9.1: protein-protein interaction networks, with increased coverage and integration.

    PubMed

    Franceschini, Andrea; Szklarczyk, Damian; Frankild, Sune; Kuhn, Michael; Simonovic, Milan; Roth, Alexander; Lin, Jianyi; Minguez, Pablo; Bork, Peer; von Mering, Christian; Jensen, Lars J

    2013-01-01

    Complete knowledge of all direct and indirect interactions between proteins in a given cell would represent an important milestone towards a comprehensive description of cellular mechanisms and functions. Although this goal is still elusive, considerable progress has been made-particularly for certain model organisms and functional systems. Currently, protein interactions and associations are annotated at various levels of detail in online resources, ranging from raw data repositories to highly formalized pathway databases. For many applications, a global view of all the available interaction data is desirable, including lower-quality data and/or computational predictions. The STRING database (http://string-db.org/) aims to provide such a global perspective for as many organisms as feasible. Known and predicted associations are scored and integrated, resulting in comprehensive protein networks covering >1100 organisms. Here, we describe the update to version 9.1 of STRING, introducing several improvements: (i) we extend the automated mining of scientific texts for interaction information, to now also include full-text articles; (ii) we entirely re-designed the algorithm for transferring interactions from one model organism to the other; and (iii) we provide users with statistical information on any functional enrichment observed in their networks.

  14. Inhibitors of cholesterol biosynthesis increase hepatic low-density lipoprotein receptor protein degradation.

    PubMed

    Ness, G C; Zhao, Z; Lopez, D

    1996-01-15

    Inhibitors of cholesterol biosynthesis are believed to lower serum cholesterol levels by enhancing the removal of serum low-density lipoprotein (LDL) by increasing hepatic LDL receptor function. Thus, the effects of several different inhibitors of cholesterol biosynthesis were examined for their effects on the expression of the hepatic LDL receptor in rats. We found that administration of inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase such as lovastatin, pravastatin, fluvastatin, and rivastatin resulted in increased hepatic LDL receptor mRNA levels. Surprisingly, these agents failed to increase levels of immunoreactive LDL receptor protein in rat liver even when the dose and length of treatment were increased. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, caused even greater increases in hepatic LDL receptor mRNA levels, but did not increase levels of immunoreactive protein. Further investigation revealed that the rate of degradation of the hepatic LDL receptor was increased in rats given inhibitors of cholesterol biosynthesis. The greatest increase in the rate of degradation was seen in animals treated with zaragozic acid A which caused the largest increase in hepatic LDL receptor mRNA levels. In contrast, hepatic LDL receptor protein was stabilized in cholesterol-fed rats. It appears that increased potential for LDL receptor protein synthesis, reflected in increased mRNA levels, is offset by a corresponding increase in the rate of receptor protein degradation resulting in constant steady-state levels of hepatic LDL receptor protein. These findings are suggestive of increased cycling of the hepatic LDL receptor. This postulated mechanism can provide for enhanced hepatic uptake of lipoproteins without increasing steady-state levels of LDL receptor protein.

  15. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  16. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    PubMed

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  17. Protein Ingestion before Sleep Increases Overnight Muscle Protein Synthesis Rates in Healthy Older Men: A Randomized Controlled Trial.

    PubMed

    Kouw, Imre Wk; Holwerda, Andrew M; Trommelen, Jorn; Kramer, Irene Fleur; Bastiaanse, Jacqueline; Halson, Shona L; Wodzig, Will Kwh; Verdijk, Lex B; van Loon, Luc Jc

    2017-08-30

    Background: The loss of skeletal muscle mass with aging has been attributed to the blunted anabolic response to protein intake. Presleep protein ingestion has been suggested as an effective strategy to compensate for such anabolic resistance.Objective: We assessed the efficacy of presleep protein ingestion on dietary protein digestion and absorption kinetics and overnight muscle protein synthesis rates in older men.Methods: In a randomized, double-blind, parallel design, 48 older men (mean ± SEM age: 72 ± 1 y) ingested 40 g casein (PRO40), 20 g casein (PRO20), 20 g casein plus 1.5 g leucine (PRO20+LEU), or a placebo before sleep. Ingestion of intrinsically l-[1-(13)C]-phenylalanine- and l-[1-(13)C]-leucine-labeled protein was combined with intravenous l-[ring-(2)H5]-phenylalanine and l-[1-(13)C]-leucine infusions during sleep. Muscle and blood samples were collected throughout overnight sleep.Results: Exogenous phenylalanine appearance rates increased after protein ingestion, but to a greater extent in PRO40 than in PRO20 and PRO20+LEU (P < 0.05). Overnight myofibrillar protein synthesis rates (based on l-[ring-(2)H5]-phenylalanine) were 0.033% ± 0.002%/h, 0.037% ± 0.003%/h, 0.039% ± 0.002%/h, and 0.044% ± 0.003%/h in placebo, PRO20, PRO20+LEU, and PRO40, respectively, and were higher in PRO40 than in placebo (P = 0.02). Observations were similar based on l-[1-(13)C]-leucine tracer (placebo: 0.047% ± 0.004%/h and PRO40: 0.058% ± 0.003%/h, P = 0.08). More protein-derived amino acids (l-[1-(13)C]-phenylalanine) were incorporated into myofibrillar protein in PRO40 than in PRO20 (0.033 ± 0.002 and 0.019 ± 0.002 MPE, respectively, P < 0.001) and tended to be higher than in PRO20+LEU (0.025 ± 0.002 MPE, P = 0.06).Conclusions: Protein ingested before sleep is properly digested and absorbed throughout the night, providing precursors for myofibrillar protein synthesis during sleep in healthy older men. Ingestion of 40 g protein before sleep increases myofibrillar

  18. Protein intake induced an increase in exercise stimulated fat oxidation during stable body weight.

    PubMed

    Soenen, Stijn; Plasqui, Guy; Smeets, Astrid J; Westerterp-Plantenga, Margriet S

    2010-12-02

    Protein-rich weight-loss diets spare fat-free mass at the cost of fat mass. The objective was to examine if there is a change in stimulated fat oxidation related to protein intake during stable body weight. Subjects' (BMI 22±2kg/m(2), age 25±8 years) maximal fat oxidation (Fat(max)) was assessed during a graded bicycle test, before and after a 3-month dietary-intervention of 2MJ/day supplements exchanged with 2MJ/d of habitual energy intake. The parallel design consisted of protein-rich supplements in the protein group and an isocaloric combination of carbohydrate and fat supplements in the control group. Daily protein intake was determined according to 24-h urine nitrogen. Body composition was measured according to a 4-compartment model by a combination of underwater-weighing technique, deuterium-dilution technique and whole-body dual-energy X-ray absorptiometry (DXA). Subjects were weight stable and did not change their physical activity. The protein group (n=12) increased protein intake (11±14g, P<0.05) and had significantly higher daily protein intake vs. control (n=4) (80±21 vs.59±11g, P<0.05). Fat(max) increased significantly in the protein group (0.08±0.08g/min, P<0.01). Fat-free mass increased independent of change in body weight (P<0.01), and fat mass and fat percentage decreased (P<0.05). Change in Fat(max) was a function of change in protein intake (r=0.623, P<0.05), and not of changes in body composition or VO(2)max. Increased stimulated fat oxidation was related to increased protein intake. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Relating the effects of protein type and content in increased-protein cheese pies to consumers' perception of satiating capacity.

    PubMed

    Marcano, J; Varela, P; Fiszman, S

    2015-02-01

    Since proteins have been shown to have the highest satiation-inducing effects of all the macronutrients, increasing the protein level is one of the main strategies for designing foods with enhanced satiating capacity. However, few studies analyze the effect that protein addition has on the texture and flavor characteristics of the target food item to relate it to the expected satiating capacity it elicits. The present work studied cheese pies with three levels of soy and whey proteins. Since the protein level altered the rheological behavior of the batters before baking and the texture of the baked pies, the feasibility of adding several protein levels for obtaining a range of final products was investigated. A check-all-that-apply questionnaire containing 32 sensory and non-sensory characteristics of the samples was given to consumers (n = 131) who also scored the perceived samples' satiating capacity. The results showed that the type and content of protein contributed distinctive sensory characteristics to the samples that could be related to their satiating capacity perception. Harder and drier samples (high protein levels) were perceived as more satiating with less perceptible sweet and milky cheese pie characteristic flavors. Soy contributed an off-flavour. These results will contribute to a better understanding of the interrelation of all these factors, aiding the development of highly palatable solid foods with enhanced satiating capacities.

  20. Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep.

    PubMed

    Brown, Laura D; Rozance, Paul J; Thorn, Stephanie R; Friedman, Jacob E; Hay, William W

    2012-08-01

    Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.

  1. Dexamethasone increases aquaporin-2 protein expression in ex vivo inner medullary collecting duct suspensions

    PubMed Central

    Chen, Minguang; Cai, Hui; Klein, Janet D.; Laur, Oskar; Chen, Guangping

    2015-01-01

    Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension. PMID:26578982

  2. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    PubMed

    Can, Özge; Holland, Nolan B

    2013-12-03

    Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of <1 mM. To elucidate the basis of this increase, the data were fit to a multidomain protein adsorption model based on the classical Langmuir isotherm. Fits of the data to the modified isotherms yield values for the equilibrium binding constants for the adsorption of AFP to ice and indicate that protein surface coverage is proportional to thermal hysteresis activity.

  3. Increasing Protein Distribution Has No Effect on Changes in Lean Mass During a Rugby Preseason.

    PubMed

    MacKenzie-Shalders, Kristen L; King, Neil A; Byrne, Nuala M; Slater, Gary J

    2016-02-01

    Increasing the frequency of protein consumption is recommended to stimulate muscle hypertrophy with resistance exercise. This study manipulated dietary protein distribution to assess the effect on gains in lean mass during a rugby preseason. Twenty-four developing elite rugby athletes (age 20.1 ± 1.4 years, mass 101.6 ± 12.0 kg; M ± SD) were instructed to consume high biological value (HBV) protein at their main meals and immediately after resistance exercise while limiting protein intake between meals. To manipulate protein intake frequency, the athletes consumed 3 HBV liquid protein supplements (22 g protein) either with main meals (bolus condition) or between meals (frequent condition) for 6 weeks in a 2 × 2 crossover design. Dietary intake and change in lean mass values were compared between conditions by analysis of covariance and correlational analysis. The dietary manipulation successfully altered the protein distribution score (average number of eating occasions containing > 20 g of protein) to 4.0 ± 0.8 and 5.9 ± 0.7 (p < .01) for the bolus and frequent conditions, respectively. There was no difference in gains in lean mass between the bolus (1.4 ± 1.5 kg) and frequent (1.5 ± 1.4 kg) conditions (p = .91). There was no clear effect of increasing protein distribution from approximately 4-6 eating occasions on changes in lean mass during a rugby preseason. However, other dietary factors may have augmented adaptation.

  4. Aerobic fitness does not modulate protein metabolism in response to increased exercise: a controlled trial

    USDA-ARS?s Scientific Manuscript database

    Purpose: This study examined how a sudden increase in exercise energy expenditure affected whole body protein turnover and nitrogen balance in people of differing aerobic fitness. We hypothesized that whole-body protein turnover would be attenuated, and nitrogen balance would be preserved, in aerobi...

  5. Protein interaction evolution from promiscuity to specificity with reduced flexibility in an increasingly complex network

    PubMed Central

    Alhindi, T.; Zhang, Z.; Ruelens, P.; Coenen, H.; Degroote, H.; Iraci, N.; Geuten, K.

    2017-01-01

    A key question regarding protein evolution is how proteins adapt to the dynamic environment in which they function and how in turn their evolution shapes the protein interaction network. We used extant and resurrected ancestral plant MADS-domain transcription factors to understand how SEPALLATA3, a protein with hub and glue properties, evolved and takes part in network organization. Although the density of dimeric interactions was saturated in the network, many new interactions became mediated by SEPALLATA3 after a whole genome triplication event. By swapping SEPALLATA3 and its ancestors between dimeric networks of different ages, we found that the protein lost the capacity of promiscuous interaction and acquired specificity in evolution. This was accompanied with constraints on conformations through proline residue accumulation, which made the protein less flexible. SHORT VEGETATIVE PHASE on the other hand (non-hub) was able to gain protein-protein interactions due to a C-terminal domain insertion, allowing for a larger interaction interface. These findings illustrate that protein interaction evolution occurs at the level of conformational dynamics, when the binding mechanism concerns an induced fit or conformational selection. Proteins can evolve towards increased specificity with reduced flexibility when the complexity of the protein interaction network requires specificity. PMID:28337996

  6. Effect of increased protein intake on renal acid load and renal hemodynamic responses.

    PubMed

    Teunissen-Beekman, Karianna F M; Dopheide, Janneke; Geleijnse, Johanna M; Bakker, Stephan J L; Brink, Elizabeth J; de Leeuw, Peter W; van Baak, Marleen A

    2016-03-01

    Increased protein intake versus maltodextrin intake for 4 weeks lowers blood pressure. Concerns exist that high-protein diets reduce renal function. Effects of acute and 4-week protein intake versus maltodextrin intake on renal acid load, glomerular filtration rate and related parameters were compared in this study. Seventy-nine overweight individuals with untreated elevated blood pressure and normal kidney function were randomized to consume a mix of protein isolates (60 g/day) or maltodextrin (60 g/day) for 4 weeks in energy balance. Twenty-four-hour urinary potential renal acid load (uPRAL) was compared between groups. A subgroup (maltodextrin N = 27, protein mix N = 25) participated in extra test days investigating fasting levels and postprandial effects of meals supplemented with a moderate protein- or maltodextrin-load on glomerular filtration rate, effective renal plasma flow, plasma renin, aldosterone, pH, and bicarbonate. uPRAL was significantly higher in the protein group after 4 weeks (P ≤ 0.001). Postprandial filtration fraction decreased further after the protein-supplemented breakfast than after the maltodextrin-supplemented breakfast after 4 weeks of supplementation (P ≤ 0.001). Fasting and postprandial levels of glomerular filtration rate, effective renal plasma flow, renin, aldosterone, angiotensin-converting enzyme, pH and bicarbonate did not differ between groups. In conclusion, 4 weeks on an increased protein diet (25% of energy intake) increased renal acid load, but did not affect renal function. Postprandial changes, except for filtration fraction, also did not differ between groups. These data suggest that a moderate increase in protein intake by consumption of a protein mix for 4 weeks causes no (undesirable) effects on kidney function in overweight and obese individuals with normal kidney function.

  7. A novel method for increasing the expression level of recombinant proteins.

    PubMed

    Wang, Aijun; Clapper, Jonathan; Guderian, Jeffery A; Foy, Teresa M; Fanger, Gary R; Retter, Marc W; Skeiky, Yasir A W

    2003-07-01

    Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

  8. Corticosteroids increase protein breakdown and loss in newly diagnosed pediatric Crohn disease.

    PubMed

    Steiner, Steven J; Noe, Joshua D; Denne, Scott C

    2011-11-01

    Children with Crohn disease have altered growth and body composition. Previous studies have demonstrated decreased protein breakdown after either corticosteroid or anti-TNF-α therapy. The aim of this study was to evaluate whole body protein metabolism during corticosteroid therapy in children with newly diagnosed Crohn disease. Children with suspected Crohn disease and children with abdominal symptoms not consistent with Crohn disease underwent outpatient metabolic assessment. Patients diagnosed with Crohn disease and prescribed corticosteroid therapy returned in 2 wk for repeat metabolic assessment. Using the stable isotopes [d5] phenylalanine, [1-(13)C] leucine, and [(15)N(2)] urea, protein kinetics were determined in the fasting state. Thirty-one children (18 controls and 13 newly diagnosed with Crohn disease) completed the study. There were no significant differences in protein breakdown or loss between patients with Crohn disease at diagnosis and controls. After corticosteroid therapy in patients with Crohn disease, the rates of appearance of phenylalanine (32%) and leucine (26%) increased significantly, reflecting increased protein breakdown, and the rate of appearance of urea also increased significantly (273%), reflecting increased protein loss. Whole body protein breakdown and loss increased significantly after 2 wk of corticosteroid therapy in children with newly diagnosed Crohn disease, which may have profound effects on body composition.

  9. Increase in local protein concentration by field-inversion gel electrophoresis.

    PubMed

    Tsai, Henghang; Leung, Hon-Chiu Eastwood

    2012-01-01

    Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and nonspecific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein recovery efficiency. Here, we describe the enhancement of protein separation efficiency up to twofold in conventional one-dimensional PAG electrophoresis (1D PAGE), two-dimensional (2D) PAGE, and native PAGE by implementing pulses of inverted electric field during gel electrophoresis.

  10. The Increasing Impact of Activity-Based Protein Profiling in Plant Science.

    PubMed

    Morimoto, Kyoko; van der Hoorn, Renier A L

    2016-03-01

    The active proteome dictates plant physiology. Yet, active proteins are difficult to predict based on transcript or protein levels, because protein activities are regulated post-translationally in their microenvironments. Over the past 10 years, activity-based protein profiling (ABPP) is increasingly used in plant science. ABPP monitors the activities of hundreds of plant proteins using tagged chemical probes that react with the active site of proteins in a mechanism-dependent manner. Since labeling is covalent and irreversible, labeled proteins can be detected and identified on protein gels and by mass spectrometry using tagged fluorophores and/or biotin. Here, we discuss general concepts, approaches and practical considerations of ABPP, before we summarize the discoveries made using 40 validated probes representing 14 chemotypes that can monitor the active state of >4,500 plant proteins. These discoveries and new opportunities indicate that this emerging functional proteomic technology is a powerful discovery tool that will have an increasing impact on plant science. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Increasing protein stability: Importance of ΔCp and the denatured state

    PubMed Central

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick

    2010-01-01

    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the β-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, ΔCp, by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles. PMID:20340133

  12. Increasing protein stability: importance of DeltaC(p) and the denatured state.

    PubMed

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick

    2010-05-01

    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the beta-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28 degrees C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, DeltaC(p), by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles.

  13. Protein Malnutrition During Juvenile Age Increases Ileal and Colonic Permeability in Rats.

    PubMed

    Eyzaguirre-Velásquez, Johana; Olavarría-Ramírez, Loreto; González-Arancibia, Camila; Díaz-Merino, Camila; Ariz, Raúl; López, Silvana; Quiroz, Waldo; Beltrán, Caroll J; Bravo, Javier A; Julio-Pieper, Marcela

    2017-05-01

    Protein malnutrition can lead to morphological and functional changes in jejunum and ileum, affecting permeability to luminal contents. Regarding the large intestine, data are scarce, especially at juvenile age. We investigated whether low-protein (LP) diet could modify ileal and colonic permeability and epithelial morphology in young rats. Isocaloric diets containing 26% (control diet) or 4% protein were given to male rats between postnatal days 40 and 60. LP-diet animals failed to gain weight and displayed decreased plasma zinc levels (a marker of micronutrient deficiency). In addition, transepithelial electrical resistance and occludin expression were reduced in their ileum and colon, indicating increased gut permeability. Macromolecule transit was not modified. Finally, LP diet induced shortening of colonic crypts without affecting muscle thickness. These data show that protein malnutrition increases not only ileum but also colon permeability in juvenile rats. Enhanced exposure to colonic luminal entities may be an additional component in the pathophysiology of protein malnutrition.

  14. Flight at low ambient humidity increases protein catabolism in migratory birds.

    PubMed

    Gerson, Alexander R; Guglielmo, Christopher G

    2011-09-09

    Although fat is the primary fuel for migratory flight in birds, protein is also used. Catabolism of tissue protein yields five times as much water per kilojoule as fat, and so one proposed function of protein catabolism is to maintain water balance during nonstop flights. To test the protein-for-water hypothesis, we flew Swainson's thrushes (Catharus ustulatus) in a climatic wind tunnel under high- and low-humidity conditions at 18°C for up to 5 hours. Flight under dry conditions increased the rates of lean mass loss and endogenous water production and also increased plasma uric acid concentration. These data demonstrate that atmospheric humidity influences fuel composition in flight and suggest that protein deposition and catabolism during migration are, in part, a metabolic strategy to maintain osmotic homeostasis during flight.

  15. Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

    PubMed Central

    Sommer, Maik S.; Schleiff, Enrico

    2014-01-01

    With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity. PMID:25085907

  16. Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor*

    PubMed Central

    Lalić, Jasna; Posavec Marjanović, Melanija; Palazzo, Luca; Perina, Dragutin; Sabljić, Igor; Žaja, Roko; Colby, Thomas; Pleše, Bruna; Halasz, Mirna; Jankevicius, Gytis; Bucca, Giselda; Ahel, Marijan; Matić, Ivan; Ćetković, Helena; Luić, Marija; Mikoč, Andreja; Ahel, Ivan

    2016-01-01

    ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor. This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism. PMID:27634042

  17. Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability.

    PubMed

    Fraser, Nicholas J; Liu, Jian-Wei; Mabbitt, Peter D; Correy, Galen J; Coppin, Chris W; Lethier, Mathilde; Perugini, Matthew A; Murphy, James M; Oakeshott, John G; Weik, Martin; Jackson, Colin J

    2016-06-05

    Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the αE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcαE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcαE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  19. Increased rates of protein evolution and asymmetric deceleration after the whole-genome duplication in yeasts.

    PubMed

    Ascencio, Diana; Ochoa, Soledad; Delaye, Luis; DeLuna, Alexander

    2017-02-06

    Whole-genome duplication (WGD) events have shaped the genomes of eukaryotic organisms. Relaxed selection after duplication along with inherent functional constraints are thought to determine the fate of the paralogs and, ultimately, the evolution of gene function. Here, we investigated the rate of protein evolution (as measured by dN/dS ratios) before and after the WGD in the hemiascomycete yeasts, and the way in which changes in such rates relate to molecular and biological function. For most groups of orthologous genes (81%) we observed a change in the rates of evolution after genome duplication. Genes with atypically-low dN/dS ratio before the WGD were prone to increase their rates of evolution after duplication. Importantly, the paralogs were often different in their rates of evolution after the WGD (50% cases), however, this was more consistent with an asymmetric deceleration in the protein-evolution rates, rather than an asymmetric increase of the initial rates. Functional-category analysis showed that regulatory proteins such as protein kinases and transcription factors were enriched in genes that increase their rates of evolution after the WGD. While changes in the rate of protein-sequence evolution were associated to protein abundance, content of disordered regions, and contribution to fitness, these features were an attribute of specific functional classes. Our results indicate that strong purifying selection in ancestral pre-duplication sequences is a strong predictor of increased rates after the duplication in yeasts and that asymmetry in evolution rate is established during the deceleration phase. In addition, changes in the rates at which paralogous sequences evolve before and after WGD are different for specific protein functions; increased rates of protein evolution after duplication occur preferentially in specific protein functions.

  20. The effect of elastomer chain flexibility on protein adsorption.

    PubMed

    Vyner, Moira C; Liu, Lina; Sheardown, Heather D; Amsden, Brian G

    2013-12-01

    Cells are known to respond differently when grown on materials of varying stiffness. However, the mechanism by which a cell senses substrate stiffness is unknown. Lower crosslink density elastomers formed from acrylated star-poly(d,l lactide-co-ϵ-caprolactone) have previously been shown to support higher smooth muscle cell proliferation in in vitro culture. This difference in growth was hypothesized to be due to differences in protein adsorption that resulted from differences in polymer chain mobility at the surface. Therefore, layer mass and viscoelastic properties were measured for HSA, IgG, fibronectin, vitronectin, and serum supplemented media adsorbed to elastomers of two crosslink densities. Significantly more fibronectin adsorbed to the lower crosslink density surface while significantly more IgG adsorbed to the higher crosslink density surface. Furthermore, differences in fibronectin and IgG layer shear moduli were observed, suggesting that there was a difference in the conformation of the adsorbed protein. ATR-FTIR analysis showed that the lower crosslink density elastomer absorbed more surface water. The increased amount of water may cause greater entropic gains upon protein adsorption to the lower crosslink density surface, which increases total protein adsorption from serum and may cause differences in protein conformation and thus cell behavior.

  1. Bactericidal Permeability Increasing Protein Gene Polymorphism is Associated with Inflammatory Bowel Diseases in the Turkish Population

    PubMed Central

    Can, Güray; Akın, Hakan; Özdemir, Filiz T.; Can, Hatice; Yılmaz, Bülent; Eren, Fatih; Atuğ, Özlen; Ünsal, Belkıs; Hamzaoğlu, Hülya O.

    2015-01-01

    Background/Aims: Inflammatory bowel disease, a chronic inflammatory disease with unknown etiology, affects the small and large bowel at different levels. It is increasingly considered that innate immune system may have a central position in the pathogenesis of the disease. As a part of the innate immune system, bactericidal permeability increasing protein has an important role in the recognition and neutralization of gram-negative bacteria. The aim of our study was to investigate the involvement of bactericidal permeability increasing protein gene polymorphism (bactericidal permeability increasing protein Lys216Glu) in inflammatory bowel disease in a large group of Turkish patients. Patients and Methods: The present study included 528 inflammatory bowel disease patients, 224 with Crohn's disease and 304 with ulcerative colitis, and 339 healthy controls. Results: Bactericidal permeability increasing protein Lys216Glu polymorphism was found to be associated with both Crohn's disease and ulcerative colitis (P = 0.0001). The frequency of the Glu/Glu genotype was significantly lower in patients using steroids and in those with steroid dependence (P = 0.012, OR, 0.80; 95% confidence interval [CI]: 0.68-0.94; P = 0.0286, OR, 0.75; 95% CI: 0.66-0.86, respectively). There was no other association between bactericidal permeability increasing protein gene polymorphism and phenotypes of inflammatory bowel disease. Conclusions: Bactericidal permeability increasing protein Lys216Glu polymorphism is associated with both Crohn's disease and ulcerative colitis. This is the first study reporting the association of bactericidal permeability increasing protein gene polymorphism with steroid use and dependence in Crohn's disease. PMID:26228368

  2. Expression of G-Protein Subunit α-14 Is Increased in Human Placentas from Preeclamptic Pregnancies

    PubMed Central

    Zhao, Ying-Jie; Zou, Qing-Yun; Li, Yan; Li, Hui-Hui; Wu, Yan-Ming; Li, Xing-Fu; Wang, Kai

    2014-01-01

    G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders. PMID:24423937

  3. Expression of G-protein subunit α-14 is increased in human placentas from preeclamptic pregnancies.

    PubMed

    Zhao, Ying-Jie; Zou, Qing-Yun; Li, Yan; Li, Hui-Hui; Wu, Yan-Ming; Li, Xing-Fu; Wang, Kai; Zheng, Jing

    2014-05-01

    G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.

  4. Increasing Fragmentation of Disulfide-Bonded Proteins for Top-Down Mass Spectrometry by Supercharging

    PubMed Central

    Zhang, Jiang; Ogorzalek Loo, Rachel R.; Loo, Joseph A.

    2015-01-01

    The disulfide bond is an important post-translational modification to form and maintain the native structure and biological functions of proteins. Characterization of disulfide bond linkages is therefore of essential interest in the structural elucidation of proteins. Top-down mass spectrometry (MS) of disulfide-bonded proteins has been hindered by relatively low sequence coverage due to disulfide cross-linking. In this study, we employed top-down ESI-MS with Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD) to study the fragmentation of supercharged proteins with multiple intramolecular disulfide bonds. With charge enhancement upon the addition of sulfolane to the analyte solution, improved protein fragmentation and disulfide bond cleavage efficiency was observed for proteins including bovine β-lactoglobulin, soybean trypsin inhibitor, human proinsulin, and chicken lysozyme. Both the number and relative abundances of product ions representing disulfide cleavage increase with increasing charge states for the proteins studied. Our studies suggest supercharging ESI-MS is a promising tool to aid in the top-down MS analysis of disulfide-bonded proteins, providing potentially useful information for the determination of disulfide bond linkages. PMID:26028988

  5. Optogenetic Stimulation Increases Level of Antiapoptotic Protein Bcl-xL in Neurons.

    PubMed

    Lanshakov, D A; Drozd, U S; Dygalo, N N

    2017-03-01

    The antiapoptotic protein Bcl-xL is associated with several neuroplastic processes such as formation of synapses, regulation of spontaneous and evoked synaptic responses, and release of neurotransmitters. Dependence of expression on activity of neurons is characteristic for many proteins participating in regulation of neuroplasticity. Whether such property is exhibited by the Bcl-xL protein was analyzed using in vivo optogenetic stimulation of hippocampal glutamatergic neurons expressing channelrhodopsin ChR2H134 under CAMKIIa promoter in the adeno-associated viral vector, followed by immunohistochemical determination of the level of Bcl-xL protein in these neurons and surrounding cells. Increase in the level of early response c-Fos protein following illumination with blue light was indicative of activation of these hippocampal neurons. The optogenetic activation of hippocampus resulted in a significant increase in the level of antiapoptotic protein Bcl-xL in the photosensitive neurons as well as in the surrounding cells. The dependence of the level of expression of Bcl-xL protein on the activity of neurons indicates that this protein possesses one more important property that is essential for participation in neuroplastic processes in the brain.

  6. High dietary protein intake is associated with an increased body weight and total death risk.

    PubMed

    Hernández-Alonso, Pablo; Salas-Salvadó, Jordi; Ruiz-Canela, Miguel; Corella, Dolores; Estruch, Ramón; Fitó, Montserrat; Arós, Fernando; Gómez-Gracia, Enrique; Fiol, Miquel; Lapetra, José; Basora, Josep; Serra-Majem, Lluis; Muñoz, Miguel Ángel; Buil-Cosiales, Pilar; Saiz, Carmen; Bulló, Mònica

    2016-04-01

    High dietary protein diets are widely used to manage overweight and obesity. However, there is a lack of consensus about their long-term efficacy and safety. Therefore, the aim of this study was to assess the effect of long-term high-protein consumption on body weight changes and death outcomes in subjects at high cardiovascular risk. A secondary analysis of the PREDIMED trial was conducted. Dietary protein was assessed using a food-frequency questionnaire during the follow-up. Cox proportional hazard models were used to estimate the multivariate-adjusted hazard ratio (HR) and 95% confidence intervals (95%CI) for protein intake in relation to the risk of body weight and waist circumference changes, cardiovascular disease, cardiovascular death, cancer death and total death. Higher total protein intake, expressed as percentage of energy, was significantly associated with a greater risk of weight gain when protein replaced carbohydrates (HR: 1.90; 95%CI: 1.05, 3.46) but not when replaced fat (HR: 1.69; 95%CI: 0.94, 3.03). However, no association was found between protein intake and waist circumference. Contrary, higher total protein intake was associated with a greater risk of all-cause death in both carbohydrate and fat substitution models (HR: 1.59; 95%CI: 1.08, 2.35; and HR: 1.66; 95%CI: 1.13, 2.43, respectively). A higher consumption of animal protein was associated with an increased risk of fatal and non-fatal outcomes when protein substituted carbohydrates or fat. Higher dietary protein intake is associated with long-term increased risk of body weight gain and overall death in a Mediterranean population at high cardiovascular risk. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  7. Pokeweed antiviral protein increases HIV-1 particle infectivity by activating the cellular mitogen activated protein kinase pathway.

    PubMed

    Mansouri, Sheila; Kutky, Meherzad; Hudak, Katalin A

    2012-01-01

    Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase that exhibits antiviral activity against several viruses. The enzyme removes purine bases from the messenger RNAs of the retroviruses Human immunodeficiency virus-1 and Human T-cell leukemia virus-1. This depurination reduces viral protein synthesis by stalling elongating ribosomes at nucleotides with a missing base. Here, we transiently expressed PAP in cells with a proviral clone of HIV-1 to examine the effect of the protein on virus production and quality. PAP reduced virus production by approximately 450-fold, as measured by p24 ELISA of media containing virions, which correlated with a substantial decline in virus protein synthesis in cells. However, particles released from PAP-expressing cells were approximately 7-fold more infectious, as determined by single-cycle infection of 1G5 cells and productive infection of MT2 cells. This increase in infectivity was not likely due to changes in the processing of HIV-1 polyproteins, RNA packaging efficiency or maturation of virus. Rather, expression of PAP activated the ERK1/2 MAPK pathway to a limited extent, resulting in increased phosphorylation of viral p17 matrix protein. The increase in infectivity of HIV-1 particles produced from PAP-expressing cells was compensated by the reduction in virus number; that is, virus production decreased upon de novo infection of cells over time. However, our findings emphasize the importance of investigating the influence of heterologous protein expression upon host cells when assessing their potential for antiviral applications.

  8. Heat-shock protein ClpL/HSP100 increases penicillin tolerance in Streptococcus pneumoniae.

    PubMed

    Tran, Thao Dang-Hien; Kwon, Hyog-Young; Kim, Eun-Hye; Kim, Ki-Woo; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

    2011-01-01

    Penicillin resistance and tolerance has been an increasing threat to the treatment of pneumococcal pneumoniae. However, no penicillin tolerance-related genes have been claimed. Here we show that a major heat shock protein ClpL/HSP100 could modulate the expression of a cell wall synthesis enzyme PBP2x, and subsequently increase cell wall thickness and penicillin tolerance in Streptococus pneumoniae.

  9. Increased temporal cortex ER stress proteins in depressed subjects who died by suicide.

    PubMed

    Bown, C; Wang, J F; MacQueen, G; Young, L T

    2000-03-01

    Regulation of ER stress proteins, such as the 78-kilodalton glucose regulated protein (GRP78) by chronic treatment with mood stabilizing drugs suggests that this family of proteins may be involved in the pathophysiology of mood disorders. Indeed, increased levels of GRP78, GRP94, and calreticulin, a third member of the ER stress protein family, were found in temporal cortex of subjects with major depressive disorder who died by suicide compared with controls and subjects who died by other means. No such differences were found in subjects with other psychiatric disorders such as bipolar disorder or schizophrenia. These data suggest a potential role for ER stress proteins in severe depression that merits further study.

  10. Integrated Management Strategies Increase Cottonseed, Oil and Protein Production: The Key Role of Carbohydrate Metabolism

    PubMed Central

    Yang, Hongkun; Zhang, Xinyue; Chen, Binglin; Meng, Yali; Wang, Youhua; Zhao, Wenqing; Zhou, Zhiguo

    2017-01-01

    Cottonseed, oil, and protein, as the by-products of cotton production, have the potential to provide commodities to meet the increasing demand of renewable bio-fuels and ruminant feed. An increase in crop yield per unit area requires high-yielding cultivar management with an economic nitrogen (N) rate, an optimal N application schedule, high-yielding plant populations and strong seedlings. Whether the integration of these agronomic practices into a coherent management system can increase the productivity of cotton fiber, embryo oil and protein requires experimental elucidation. In this 2-year study, conventional management practices (CM) were used as a control, and two integrated management strategies (IMS1 and IMS2) were considered at two soil fertility levels (high soil fertility and low soil fertility) to analyze the metabolic and biochemical traits of cotton embryos. The results illustrate that the cottonseed, oil, and protein yields for IMS1 and IMS2 were significantly higher than those under CM at both soil fertility levels and the fiber yield increased as well. The IMS regulated the maternal photo thermal environment by delaying the flowering date, resulting in increases in the seed weight. In developing cotton embryos, the IMS increased the embryo weight accumulation rate and biomass partitioning into oil and protein, which were associated with high activities of H+-ATPase, H+-PPase, sucrose synthase (SuSy), and cell wall invertase (C-INV) and low activities of sucrose phosphate synthase (SPS) and vacuole invertase (V-INV). Increased hexoses (D-fructose, D-glucose) content contributed to the oil and protein contents. These results suggest that increased sucrose/H+ symport, sucrose hydrolysis, hexoses synthesis, and cumulative photo-thermal product (PTP), especially in the early stage of embryo growth, play a dominant role in the high productivity of cotton oil and protein. PMID:28194156

  11. Dietary soy isoflavones increased hepatic protein disulfide isomerase content and suppressed its enzymatic activity in rats.

    PubMed

    Xiao, Chao Wu; Donak, Kevin; Ly, Olivia; Wood, Carla; Cooke, Gerard; Curran, Ivan

    2014-06-01

    Protein disulfide isomerase (PDI) is a multifunctional protein and plays important roles in protein folding, triglyceride transfer, insulin degradation, and thyroid hormone transportation. This study examined the modulation of PDI expression by soy consumption using rat as a model. Sprague-Dawley male and female rats at 50 days (d) of age were fed diets containing either 20% casein or alcohol-washed soy protein isolate (SPI, containing 50 mg isoflavones (ISFs)/kg diet) or SPI plus ISF (250 mg/kg diet) and mated at age of 120 d. The offspring (F1) were fed the same diets as their parents. Addition of ISF to SPI diet markedly increased PDI protein content in the liver and testis of the adult rats compared with the casein or SPI diet. PDI mRNA abundance in the liver and protein content in the brain, thyroid, heart, and uterus were unchanged by the diets. Two-dimensional Western blot showed that the rats fed diets containing SPI had a diminished hepatic PDI protein with an isoelectric point (pI) of 6.12, a dephosphorylated form, compared with the rats fed diets containing either casein or SPI with supplemental ISF. Soy ISF added into SPI diet remarkably suppressed hepatic PDI activity of the rats compared with the casein diet. Moreover, soy ISF dose-dependently increased PDI and thyroid hormone receptor (TR) β protein content, whereas reduced TR DNA binding ability in human hepatocytes. Overall, this study shows that soy ISF increased hepatic PDI protein content, but addition of ISF into SPI diet inhibited its enzymatic activities and this effect may be mediated through a post-transcriptional mechanism.

  12. Increasing HDL levels by inhibiting cholesteryl ester transfer protein activity in rabbits with hindlimb ischemia is associated with increased angiogenesis.

    PubMed

    Wu, Ben J; Shrestha, Sudichhya; Ong, Kwok L; Johns, Douglas; Dunn, Louise L; Hou, Liming; Barter, Philip J; Rye, Kerry-Anne

    2015-11-15

    High density lipoprotein (HDL) infusions increase new blood vessel formation (angiogenesis) in rodents with ischemic injury. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein (CETP) activity increases angiogenesis in New Zealand White (NZW) rabbits with hindlimb ischemia. NZW rabbits were maintained for 6weeks on chow or chow supplemented with 0.07% or 0.14% (wt/wt) of the CETP inhibitor, des-fluoro-anacetrapib. The left femoral artery was ligated after 2weeks of des-fluoro-anacetrapib treatment. The animals were sacrificed 4weeks after femoral artery ligation. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib reduced CETP activity by 63±12% and 81±8.6%, increased plasma apoA-I levels by 1.3±0.1- and 1.4±0.1-fold, and increased plasma HDL-cholesterol levels by 1.4±0.1- and 1.7±0.2-fold, respectively. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib increased the number of collateral arteries by 60±16% and 84±27%, and arteriole wall area in the ischemic hindlimbs by 84±16% and 94±13%, respectively. Capillary density in the ischemic hindlimb adductor muscle increased from 1.1±0.2 (control) to 2.1±0.3 and 2.2±0.4 in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively. Incubation of HDLs from des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells at apoA-I concentrations comparable with their plasma levels increased tubule network formation. These effects were abolished by knockdown of scavenger receptor-B1 (SR-B1) and PDZK1, and pharmacological inhibition of PI3K/Akt. Increasing HDL levels by inhibiting CETP activity is associated with increased collateral blood vessel formation in NZW rabbits with hindlimb ischemia in an SR-B1- and PI3K/Akt-dependent manner. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Enzymatic Pre-Treatment Increases the Protein Bioaccessibility and Extractability in Dulse (Palmaria palmata)

    PubMed Central

    Mæhre, Hanne K.; Jensen, Ida-Johanne; Eilertsen, Karl-Erik

    2016-01-01

    Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with alkaline extraction resulted in a 1.6-fold increase in the protein extraction yield compared to a standard alkaline extraction protocol. A simulated in vitro gastrointestinal digestion model showed that enzymatic pre-treatment of seaweed increased the amount of amino acids available for intestinal absorption 3.2-fold. In conclusion, enzymatic pre-treatment of seaweeds is effective for increasing the amount of amino acids available for utilization and may thus be an effective means for increasing the utilization potential of seaweed proteins. However, both the enzymatic pre-treatment protocol and the protein extraction protocol need further optimization in order to obtain optimal cost-benefit and results from the in vitro gastrointestinal digestion model need to be confirmed in clinical models. PMID:27792166

  14. Whole body and forearm substrate metabolism in hyperthyroidism: evidence of increased basal muscle protein breakdown.

    PubMed

    Riis, Anne Lene Dalkjaer; Jørgensen, Jens Otto Lunde; Gjedde, Signe; Nørrelund, Helene; Jurik, Anne Grethe; Nair, K S; Ivarsen, Per; Weeke, Jørgen; Møller, Niels

    2005-06-01

    Thyroid hormones have significant metabolic effects, and muscle wasting and weakness are prominent clinical features of chronic hyperthyroidism. To assess the underlying mechanisms, we examined seven hyperthyroid women with Graves' disease before (Ht) and after (Eut) medical treatment and seven control subjects (Ctr). All subjects underwent a 3-h study in the postabsorptive state. After regional catheterization, protein dynamics of the whole body and of the forearm muscles were measured by amino acid tracer dilution technique using [15N]phenylalanine and [2H4]tyrosine. Before treatment, triiodothyronine was elevated (6.6 nmol/l) and whole body protein breakdown was increased 40%. The net forearm release of phenylalanine was increased in hyperthyroidism (microg.100 ml(-1).min(-1)): -7.0 +/- 1.2 Ht vs. -3.8 +/- 0.8 Eut (P = 0.04), -4.2 +/- 0.3 Ctr (P = 0.048). Muscle protein breakdown, assessed by phenylalanine rate of appearance, was increased (microg.100 ml(-1).min(-1)): 15.5 +/- 2.0 Ht vs. 9.6 +/- 1.4 Eut (P = 0.03), 9.9 +/- 0.6 Ctr (P = 0.02). Muscle protein synthesis rate did not differ significantly. Muscle mass and muscle function were decreased 10-20% before treatment. All abnormalities were normalized after therapy. In conclusion, our results show that hyperthyroidism is associated with increased muscle amino acid release resulting from increased muscle protein breakdown. These abnormalities can explain the clinical manifestations of sarcopenia and myopathy.

  15. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    PubMed

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  16. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    PubMed Central

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  17. Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein.

    PubMed

    Lee, Duck Yeon; Kim, Kyeong-Ae; Yu, Yeon Gyu; Kim, Key-Sun

    2004-07-30

    Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.

  18. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  19. Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

    PubMed

    Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

    2017-09-19

    A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Overexpression of the RieskeFeS Protein Increases Electron Transport Rates and Biomass Yield.

    PubMed

    Simkin, Andrew J; McAusland, Lorna; Lawson, Tracy; Raines, Christine A

    2017-09-01

    In this study, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing the Rieske FeS protein (PetC), a component of the cytochrome b6f (cyt b6f) complex. Increasing the levels of this protein resulted in concomitant increases in the levels of cyt f (PetA) and cyt b6 (PetB), core proteins of the cyt b6f complex. Interestingly, an increase in the levels of proteins in both the photosystem I (PSI) and PSII complexes also was seen in the Rieske FeS overexpression plants. Although the mechanisms leading to these changes remain to be identified, the transgenic plants presented here provide novel tools to explore this. Importantly, overexpression of the Rieske FeS protein resulted in substantial and significant impacts on the quantum efficiency of PSI and PSII, electron transport, biomass, and seed yield in Arabidopsis plants. These results demonstrate the potential for manipulating electron transport processes to increase crop productivity. © 2017 The author(s). All Rights Reserved.

  1. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate

    PubMed Central

    Keren, Leeat; Segal, Eran; Milo, Ron

    2016-01-01

    Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

  2. Increased serum G72 protein levels in patients with schizophrenia: a potential candidate biomarker.

    PubMed

    Akyol, Esra Soydaş; Albayrak, Yakup; Aksoy, Nurkan; Şahin, Başak; Beyazyüz, Murat; Kuloğlu, Murat; Hashimoto, Kenji

    2017-04-01

    The product of the G72 gene is an activator of d-amino acid oxidase and has been suggested to play a role in the pathogenesis of schizophrenia. Increased G72 protein levels may be associated with disturbed glutamatergic transmission and increased reactive oxygen species. Only one pilot study by Lin et al. has investigated the potential role of serum G72 protein levels as a biomarker for schizophrenia. In this study, we aimed to compare serum G72 protein levels between patients with schizophrenia and healthy controls, and to retest the results of the previous pilot study. Materials and methods In total, 107 patients with a diagnosis of schizophrenia according to the inclusion and exclusion criteria and 60 age-sex-matched healthy controls were included in the study. The groups were compared regarding serum G72 protein levels. The mean serum G72 protein values were 495.90±152.03 pg/ml in the schizophrenia group and 346.10±102.08 pg/ml in the healthy control group. The mean serum G72 protein level was significantly increased in the schizophrenia group compared with the healthy control group (t=-3.89, p<0.001). A receiver operating characteristics analysis was performed to compare the schizophrenia and healthy control groups. It was determined that the cut-off value was 141.51 pg/ml with a sensitivity of 0.991 and a specificity of 0.821. We suggest that serum G72 protein levels may represent a candidate biomarker for schizophrenia and have confirmed the results of the previous preliminary study. Additional studies with larger sample sizes and the inclusion of first episode schizophrenia patients are required to clarify the reliability and validity of serum G72 protein levels as a biomarker for schizophrenia.

  3. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate.

    PubMed

    Barenholz, Uri; Keren, Leeat; Segal, Eran; Milo, Ron

    2016-01-01

    Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms.

  4. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

  5. Hierarchical protein patterning by meso to molecular scale self-assembly

    NASA Astrophysics Data System (ADS)

    Andersen, Andreas S.; Sutherland, Duncan S.; Ogaki, Ryosuke

    2015-10-01

    Numerous protein patterning methodologies are used extensively for biomedical research and development. We have developed a novel bottom-up protein patterning method using a combination of self-assembly processes in the meso to molecular scale range to allow hierarchical protein patterns to be straightforwardly fabricated with low cost over large areas. As a proof of principle, we patterned vitronectin in various dimensional hierarchies using meso to nanoscale colloids and self-assembled monolayers.

  6. Possible role for increased C4b-binding-protein level in acquired protein S deficiency in type I diabetes.

    PubMed

    Ceriello, A; Giugliano, D; Quatraro, A; Marchi, E; Barbanti, M; Lefebvre, P

    1990-04-01

    In this study, total protein S (PS) immunological levels, free-PS and C4b-binding-protein (C4bBP) concentrations, and PS functional activity were investigated in insulin-dependent (type I) diabetic patients and compared with nondiabetic subjects. Mean total PS antigen concentration was not different between diabetic patients and nondiabetic subjects, whereas free-PS levels and PS functional activity were significantly reduced in diabetic patients. C4bBP was increased in diabetic patients and correlated with HbA1 levels. This study shows that type I diabetic patients have depressed free PS and PS activity despite the presence of normal total PS concentration and suggests that this phenomenon is probably linked to the increase of circulating C4bBP.

  7. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy.

    PubMed

    Radley-Crabb, Hannah G; Marini, Juan C; Sosa, Horacio A; Castillo, Liliana I; Grounds, Miranda D; Fiorotto, Marta L

    2014-01-01

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles.

  8. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    PubMed Central

    Zhou, Yuping; Vachet, Richard W.

    2012-01-01

    Covalent labeling and mass spectrometry are seeing increased used together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g. diethylpyrocarbonate) and non-specific (e.g. hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues, and thus protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g. 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g. microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. As compared to typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 Å to 7 Å for myoglobin, 13 Å to 10 Å for

  9. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    NASA Astrophysics Data System (ADS)

    Zhou, Yuping; Vachet, Richard W.

    2012-04-01

    Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

  10. Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields.

    PubMed

    Marin Viegas, Vanesa Soledad; Ocampo, Carolina Gabriela; Petruccelli, Silvana

    2017-05-04

    Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, α1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms

  11. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Haas, A. L.; Goldberg, A. L.

    1995-01-01

    The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

  12. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Haas, A. L.; Goldberg, A. L.

    1995-01-01

    The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

  13. Salt bridges in the hyperthermophilic protein Ssh10b are resilient to temperature increases.

    PubMed

    Ge, Meng; Xia, Xia-Yu; Pan, Xian-Ming

    2008-11-14

    A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.

  14. Prebiotics affect nutrient digestibility but not faecal ammonia in dogs fed increased dietary protein levels.

    PubMed

    Hesta, M; Roosen, W; Janssens, G P J; Millet, S; De Wilde, R

    2003-12-01

    An increased protein content and less digestible protein sources in the diet can induce bad faecal odour. The present study investigated the effect of adding prebiotics to dog diets enriched with animal-derived protein sources on apparent digestibilities and faecal ammonia concentration. In three subsequent periods eight healthy beagle dogs were fed a commercial dog diet that was gradually supplemented by up to 50 % with meat and bone meal (MBM), greaves meal (GM) or poultry meal (PM) respectively. Afterwards, 3 % fructo-oligosaccharides or 3 % isomalto-oligosaccharides were substituted for 3 % of the total diet. Supplementation with animal-derived protein sources did not decrease the apparent N digestibility significantly but oligosaccharides did. On the other hand the bacterial N content (% DM) in the faeces was highest in the oligosaccharide groups followed by the protein-supplemented groups and lowest in the control groups. When the apparent N digestibility was corrected for bacterial N no significant differences were noted anymore except for the GM group where the corrected N digestibility was still lower after oligosaccharide supplementation. The amount of faecal ammonia was significantly increased by supplementing with protein or oligosaccharides in the MBM and GM groups but not in the PM group. When apparent N digestibility is interpreted, a correction for bacterial N should be taken into account, especially when prebiotics are added to the diet. Oligosaccharides did not reduce the faecal ammonia concentrations as expected.

  15. Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

    PubMed

    Conn, Crystal S; Qian, Shu-Bing

    2013-04-16

    The discovery that rapamycin extends the life span of diverse organisms has triggered many studies aimed at identifying the underlying molecular mechanisms. Mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and may regulate organismal aging by controlling mRNA translation. However, how inhibiting mTORC1 and decreasing protein synthesis can extend life span remains an unresolved issue. We showed that constitutively active mTORC1 signaling increased general protein synthesis but unexpectedly reduced the quality of newly synthesized polypeptides. We demonstrated that constitutively active mTORC1 decreased translation fidelity by increasing the speed of ribosomal elongation. Conversely, rapamycin treatment restored the quality of newly synthesized polypeptides mainly by slowing the rate of ribosomal elongation. We also found distinct roles for mTORC1 downstream targets in maintaining protein homeostasis. Loss of S6 kinases, but not 4E-BP family proteins, which are both involved in regulation of translation, attenuated the effects of rapamycin on the quality of newly translated proteins. Our results reveal a mechanistic connection between mTORC1 and protein quality, highlighting the central role of nutrient signaling in growth and aging.

  16. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    PubMed Central

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W.; Amin, Hesham M.; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D.; Rosen, Steven T.; Pham, Lan V.; Ngo, Vu N.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL. PMID:27713153

  17. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

    PubMed

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W; Amin, Hesham M; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D; Rosen, Steven T; Pham, Lan V; Ngo, Vu N

    2016-11-08

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

  18. Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts.

    PubMed

    Abraham, A K; Kolseth, S; Pihl, A

    1982-05-17

    Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.

  19. Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia.

    PubMed

    Funk, Adam J; Haroutunian, Vahram; Meador-Woodruff, James H; McCullumsmith, Robert E

    2014-10-01

    Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand-bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that the expression of GRK proteins is altered in schizophrenia, consistent with previous findings of alterations upstream and downstream from this family of molecules that facilitate intracellular signaling processes. In this study, we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (n=36) and a comparison group (n=33). To control for antipsychotic treatment, we measured these same targets in haloperidol-treated vs. untreated rats (n=10 for both). We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. These data suggest that increased GRK5 expression may contribute to the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Identification of Ubiquilin, a Novel Presenilin Interactor That Increases Presenilin Protein Accumulation

    PubMed Central

    Mah, Alex L.; Perry, George; Smith, Mark A.; Monteiro, Mervyn J.

    2000-01-01

    Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain. PMID:11076969

  1. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.

    PubMed

    Niiranen, Laila; Espelid, Sigrun; Karlsen, Christian R; Mustonen, Milla; Paulsen, Steinar M; Heikinheimo, Pirkko; Willassen, Nils P

    2007-03-01

    Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.

  2. Orally administered lactoferrin increases hepatic protein synthesis in formula-fed newborn pigs.

    PubMed

    Burrin, D G; Wang, H; Heath, J; Dudley, M A

    1996-07-01

    Lactoferrin is a polypeptide which is abundant in colostrum; however, its biologic effect in the neonate is unknown. The objective was to determine the potentially anabolic effect of orally administered lactoferrin on visceral organ growth and protein synthesis in newborn pigs. We studied a total of 18 unsuckled newborn pigs from six litters. Three pigs from each litter were randomly assigned to one of three dietary treatment groups (n = 6) and bottle-fed (10 mL/h) formula, formula containing physiologic levels (1 mg/mL) of added bovine lactoferrin (bLF), or colostrum. After 24 h of feeding, we measured visceral organ protein synthesis in vivo using a flooding dose of [3H]phenylalanine. We also measured visceral organ protein and DNA mass, as well as intestinal hydrolase activities and villus morphology. Hepatic protein synthesis in pigs fed either formula containing bLF or colostrum was similar and in both groups was significantly higher than in pigs fed formula. Splenic protein synthesis was not significantly different in pigs fed either formula or formula containing bLF, but was significantly higher in colostrum-fed animals. There were no significant differences in small intestinal growth, protein synthesis, or hydrolase activities between newborn pigs fed formula, formula containing bLF, or colostrum. Our results demonstrate that feeding formula containing physiologic concentrations of added bLF increased hepatic protein synthesis in newborn pigs, suggesting that colostrumborne lactoferrin serves an anabolic function in neonates.

  3. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  4. Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

    PubMed Central

    Wang, Hui; Zhao, Jun-Xing; Hu, Nan; Ren, Jun; Du, Min; Zhu, Mei-Jun

    2012-01-01

    AIM: To investigate the effect of side-stream smoking on gut microflora composition, intestinal inflammation and expression of tight junction proteins. METHODS: C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks. Cecal contents were collected for microbial composition analysis. Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins. RESULTS: Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria, Clostridium but decreased Fermicutes (Lactoccoci and Ruminococcus), Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice. Meanwhile, side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα, accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6. The contents of tight junction proteins, claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking. In addition, side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling, while inhibiting AMP-activated protein kinase in the large intestine. CONCLUSION: Side-stream smoking altered gut microflora composition and reduced the inflammatory response, which was associated with increased expression of tight junction proteins. PMID:22611310

  5. Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction.

    PubMed

    Ding, Ning; Yang, Chunguang; Sun, Shenxia; Han, Lichi; Ruan, Yao; Guo, Longhua; Hu, Xuejun; Zhang, Jianing

    2017-03-25

    Escherichia coli cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. coli is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto-induction procedures. To justify the potential for general application of the optimized auto-induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. coli cells.

  6. Strong Selection Significantly Increases Epistatic Interactions in the Long-Term Evolution of a Protein

    PubMed Central

    Gupta, Aditi; Adami, Christoph

    2016-01-01

    Epistatic interactions between residues determine a protein’s adaptability and shape its evolutionary trajectory. When a protein experiences a changed environment, it is under strong selection to find a peak in the new fitness landscape. It has been shown that strong selection increases epistatic interactions as well as the ruggedness of the fitness landscape, but little is known about how the epistatic interactions change under selection in the long-term evolution of a protein. Here we analyze the evolution of epistasis in the protease of the human immunodeficiency virus type 1 (HIV-1) using protease sequences collected for almost a decade from both treated and untreated patients, to understand how epistasis changes and how those changes impact the long-term evolvability of a protein. We use an information-theoretic proxy for epistasis that quantifies the co-variation between sites, and show that positive information is a necessary (but not sufficient) condition that detects epistasis in most cases. We analyze the “fossils” of the evolutionary trajectories of the protein contained in the sequence data, and show that epistasis continues to enrich under strong selection, but not for proteins whose environment is unchanged. The increase in epistasis compensates for the information loss due to sequence variability brought about by treatment, and facilitates adaptation in the increasingly rugged fitness landscape of treatment. While epistasis is thought to enhance evolvability via valley-crossing early-on in adaptation, it can hinder adaptation later when the landscape has turned rugged. However, we find no evidence that the HIV-1 protease has reached its potential for evolution after 9 years of adapting to a drug environment that itself is constantly changing. We suggest that the mechanism of encoding new information into pairwise interactions is central to protein evolution not just in HIV-1 protease, but for any protein adapting to a changing environment. PMID

  7. The Transient Nature of Bunyamwera Orthobunyavirus NSs Protein Expression: Effects of Increased Stability of NSs Protein on Virus Replication

    PubMed Central

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M.

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein. PMID:23667701

  8. Tyrosine phosphorylation plays a role in increasing maspin protein levels and its cytoplasmic accumulation

    PubMed Central

    Tamazato Longhi, Mariana; Cella, Nathalie

    2012-01-01

    Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation. PMID:23650586

  9. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    PubMed Central

    Keefe, Andrew J.; Jiang, Shaoyi

    2013-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein–substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity. PMID:22169873

  10. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain.

    PubMed

    Costa, D F A; Quigley, S P; Isherwood, P; McLennan, S R; Poppi, D P

    2016-05-01

    A series of 3 experiments were conducted to evaluate the use of microalgae as supplements for ruminants consuming low-CP tropical grasses. In Exp. 1, the chemical composition and in vitro protein degradability of 9 algae species and 4 protein supplements were determined. In Exp. 2, rumen function and microbial protein (MCP) production were determined in steers fed speargrass hay alone or supplemented with , , , or cottonseed meal (CSM). In Exp. 3, DMI and ADG were determined in steers fed speargrass hay alone or supplemented with increasing amounts of NPN (urea combined with ammonia sulfate), CSM, or . In Exp. 1, the CP content of and (675 and 580 g/kg DM) was highest among the algae species and higher than the other protein supplements evaluated, and sp. had the highest crude lipid (CL) content (198 g/kg DM). In Exp. 2, supplementation increased speargrass hay intake, the efficiency of MCP production, the fractional outflow rate of digesta from the rumen, the concentration of NHN, and the molar proportion of branched-chain fatty acids in the rumen fluid of steers above all other treatments. acceptance by steers was low and this resulted in no significant difference to unsupplemented steers for all parameters measured for this algae supplement. In Exp. 3, ADG linearly increased with increasing supplementary N intake from both and NPN, with no difference between the 2 supplements. In contrast, ADG quadratically increased with increasing supplementary N intake from CSM. It was concluded that and may potentially be used as protein sources for cattle grazing low-CP pastures.

  11. A Ketogenic Diet Increases Brown Adipose Tissue Mitochondrial Proteins and UCP1 Levels in Mice

    PubMed Central

    Srivastava, Shireesh; Baxa, Ulrich; Niu, Gang; Chen, Xiaoyuan; Veech, Richard L.

    2013-01-01

    We evaluated the effects of feeding a ketogenic diet (KD) for a month on general physiology with emphasis on brown adipose tissue (BAT) in mice. KD did not reduce the caloric intake, or weight or lipid content of BAT. Relative epididymal fat pads were 40% greater in the mice fed the KD (P = 0.06) while leptin was lower (P < 0.05). Blood glucose levels were 30% lower while D-β-hydroxybutyrate levels were about 3.5-fold higher in the KD group. Plasma insulin and leptin levels in the KD group were about half of that of the mice fed NIH-31 pellets (chow group). Median mitochondrial size in the inter-scapular BAT (IBAT) of the KD group was about 60% greater, whereas the median lipid droplet size was about half of that in the chow group. Mitochondrial oxidative phosphorylation proteins were increased (1.5–3-fold) and the uncoupling protein 1 levels were increased by threefold in mice fed the KD. The levels of PPARγ, PGC-1α, and Sirt1 in KD group were 1.5–3-fold while level of Sirt3 was about half of that in the chow-fed group. IBAT cyclic AMP levels were 60% higher in the KD group and cAMP response element binding protein was 2.5-fold higher, suggesting increased sympathetic system activity. These results demonstrate that a KD can also increase BAT mitochondrial size and protein levels. PMID:23233333

  12. Increase in bone protein components with healing rat fractures: enhancement by zinc treatment.

    PubMed

    Igarashi, A; Yamaguchi, M

    1999-12-01

    The alteration in bone components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed 7 and 14 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many protein molecules were induced in the diaphyseal tissues with fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 and 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Also, the culture of the diaphyseal tissues with fracture healing caused a significant increase in bone alkaline phosphatase activity and deoxyribonucleic acid (DNA) content. Meanwhile, the presence of zinc acexamate (10-5 and 10-4 M), a stimulator of bone formation, in a culture medium induced a significant elevation of protein content and alkaline phosphatase activity in the diaphyseal tissues with fracture healing. Such an effect was completely abolished by the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis. The present study suggests that fracture healing induces a newly synthesized bone protein component including stimulatory factor(s) for bone formation. Zinc supplementation may stimulate the healing of femoral fracture.

  13. Highly increased detection of silver stained protein bands in polyacrylamide gels with thermo-optical methods

    NASA Astrophysics Data System (ADS)

    Mazza, Giulia; Posnicek, Thomas; Brandl, Martin

    2016-11-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a well-known technique to separate proteins by their molecular weight. After electrophoresis, the gels are commonly stained for protein band analysis with silver stain; this allows the detection of protein loads to about 1 ng. To increase the detection sensitivity of the protein bands down in the subnanogram level, a sensor has been developed based on the thermal lens effect to scan and quantify protein loads which would remain undetected using the standard imaging systems. The thermal lens sensor is equipped with a 450 nm diode pump laser modulated at 1 Hz and a HeNe probe laser mounted in collinear geometry. The sensor could detect protein bands of 0.05 ng when the gel was soaked in methanol/water and 0.1 ng in water. The limit of detection ranged from 8 to 20 pg, depending on the soaking medium and the staining efficiency. Thus, the detection of silver stain by thermal lens effect results 10 to 20 times more sensitive than the standard colorimetric method.

  14. Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

    PubMed Central

    Kumar, G.N. Mohan; Houtz, Robert L.; Knowles, N. Richard

    1999-01-01

    Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers. PMID:9880350

  15. HIF1α protein stability is increased by acetylation at lysine 709.

    PubMed

    Geng, Hao; Liu, Qiong; Xue, Changhui; David, Larry L; Beer, Tomasz M; Thomas, George V; Dai, Mu-Shui; Qian, David Z

    2012-10-12

    Lysine acetylation regulates protein stability and function. p300 is a component of the HIF-1 transcriptional complex and positively regulates the transactivation of HIF-1. Here, we show a novel molecular mechanism by which p300 facilitates HIF-1 activity. p300 increases HIF-1α (HIF1α) protein acetylation and stability. The regulation can be opposed by HDAC1, but not by HDAC3, and is abrogated by disrupting HIF1α-p300 interaction. Mechanistically, p300 specifically acetylates HIF1α at Lys-709, which increases the protein stability and decreases polyubiquitination in both normoxia and hypoxia. Compared with the wild-type protein, a HIF1α K709A mutant protein is more stable, less polyubiquitinated, and less dependent on p300. Overexpression of the HIF1α wild-type or K709A mutant in cancer cells lacking the endogenous HIF1α shows that the K709A mutant is transcriptionally more active toward the HIF-1 reporter and some endogenous target genes. Cancer cells containing the K709A mutant are less sensitive to hypoxia-induced growth arrest than the cells containing the HIF1α wild-type. Taken together, these data demonstrate a novel biological consequence upon HIF1α-p300 interaction, in which HIF1α can be stabilized by p300 via Lys-709 acetylation.

  16. Age-induced protein modifications and increased proteolysis in potato seed-tubers

    SciTech Connect

    Kumar, G.N.M.; Knowles, N.R.; Houtz, R.L.

    1999-01-01

    Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as in increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed daughter tubers that contained 3-fold more protein than mother tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Their results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; the authors also identify a role for PMC in regulating protein turnover in potato tubers.

  17. Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice

    PubMed Central

    Oristian, Daniel S.; Sloofman, Laura G.; Zhou, Xiaozhou; Wang, Liyun; Farach-Carson, Mary C.; Kirn-Safran, Catherine B.

    2008-01-01

    SUMMARY Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29-null mice is due to delayed entry of chondro-osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro-computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender-specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender-matched controls in three point-bending tests, and smaller post-yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29-deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub-efficient protein translation increased fracture risk in HIP/RPL29-null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality. PMID:18661500

  18. Increased Myeloperoxidase Activity and Protein Nitration Are Indicators of Inflammation in Patients with Chagas' Disease▿

    PubMed Central

    Dhiman, Monisha; Estrada-Franco, Jose Guillermo; Pando, Jasmine M.; Ramirez-Aguilar, Francisco J.; Spratt, Heidi; Vazquez-Corzo, Sara; Perez-Molina, Gladys; Gallegos-Sandoval, Rosa; Moreno, Roberto; Garg, Nisha Jain

    2009-01-01

    In this study, we investigated whether inflammatory responses contribute to oxidative/nitrosative stress in patients with Chagas' disease. We used three tests (enzyme-linked immunosorbent assay, immuno-flow cytometry, and STAT-PAK immunochromatography) to screen human serum samples (n = 1,481) originating from Chiapas, Mexico, for Trypanosoma cruzi-specific antibodies. We identified 121 subjects who were seropositive for T. cruzi-specific antibodies, a finding indicative of an 8.5% seroprevalence in the rural population from Chiapas. Seropositive and seronegative subjects were examined for plasma levels of biomarkers of inflammation, i.e., myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and xanthine oxidase (XOD), as well as for oxidative (advanced oxidation protein products [AOPPs]) and nitrosative (3-nitrotyrosine [3NT]) biomarkers. The seropositive subjects exhibited a significant increase in MPO activity and protein level, the indicator of neutrophil activation. Subsequently, a corresponding increase in AOPP contents, formed by MPO-dependent hypochlorous acid and chloramine formation, was noted in seropositive subjects. The plasma level of 3NT was significantly increased in seropositive subjects, yet we observed no change in XOD activity (O2− source) and nitrate/nitrite contents (denotes iNOS activation and NO production), which implied that direct peroxynitrite formation does not contribute to increased nitrosative damage in chagasic subjects. Instead, a positive correlation between increased MPO activity and protein 3NT formation was observed, which suggested to us that MPO-dependent formation of nitrylchloride that occurs in the presence of physiological NO and O2− concentrations contributes to protein nitration. Overall, our data demonstrate that T. cruzi-induced neutrophil activation is pathological and contributes to MPO-mediated collateral protein oxidative and nitrosative damage in human patients with Chagas' disease. Therapies

  19. Exercise training increases protein O-GlcNAcylation in rat skeletal muscle.

    PubMed

    Hortemo, Kristin Halvorsen; Lunde, Per Kristian; Anonsen, Jan Haug; Kvaløy, Heidi; Munkvik, Morten; Rehn, Tommy Aune; Sjaastad, Ivar; Lunde, Ida Gjervold; Aronsen, Jan Magnus; Sejersted, Ole M

    2016-09-01

    Protein O-GlcNAcylation has emerged as an important intracellular signaling system with both physiological and pathophysiological functions, but the role of protein O-GlcNAcylation in skeletal muscle remains elusive. In this study, we tested the hypothesis that protein O-GlcNAcylation is a dynamic signaling system in skeletal muscle in exercise and disease. Immunoblotting showed different protein O-GlcNAcylation pattern in the prototypical slow twitch soleus muscle compared to fast twitch EDL from rats, with greater O-GlcNAcylation level in soleus associated with higher expression of the modulating enzymes O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine fructose-6-phosphate amidotransferase isoforms 1 and 2 (GFAT1, GFAT2). Six weeks of exercise training by treadmill running, but not an acute exercise bout, increased protein O-GlcNAcylation in rat soleus and EDL There was a striking increase in O-GlcNAcylation of cytoplasmic proteins ~50 kDa in size that judged from mass spectrometry analysis could represent O-GlcNAcylation of one or more key metabolic enzymes. This suggests that cytoplasmic O-GlcNAc signaling is part of the training response. In contrast to exercise training, postinfarction heart failure (HF) in rats and humans did not affect skeletal muscle O-GlcNAcylation level, indicating that aberrant O-GlcNAcylation cannot explain the skeletal muscle dysfunction in HF Human skeletal muscle displayed extensive protein O-GlcNAcylation that by large mirrored the fiber-type-related O-GlcNAcylation pattern in rats, suggesting O-GlcNAcylation as an important signaling system also in human skeletal muscle. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  20. A Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

    SciTech Connect

    Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.; Matzke, Melissa M.; Nair, Parameswaran; Bigelow, Diana J.; Pounds, Joel G.; Zangar, Richard C.

    2014-01-31

    Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, we found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from

  1. The p14 FAST Protein of Reptilian Reovirus Increases Vesicular Stomatitis Virus Neuropathogenesis▿

    PubMed Central

    Brown, Christopher W.; Stephenson, Kyle B.; Hanson, Stephen; Kucharczyk, Michael; Duncan, Roy; Bell, John C.; Lichty, Brian D.

    2009-01-01

    The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor. PMID:18971262

  2. The p14 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis.

    PubMed

    Brown, Christopher W; Stephenson, Kyle B; Hanson, Stephen; Kucharczyk, Michael; Duncan, Roy; Bell, John C; Lichty, Brian D

    2009-01-01

    The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.

  3. Heparin treatment increases thioredoxin interacting protein expression in hepatocellular carcinoma cells.

    PubMed

    Gunes, Aysim; Iscan, Evin; Topel, Hande; Avci, Sanem Tercan; Gumustekin, Mukaddes; Erdal, Esra; Atabey, Nese

    2015-08-01

    Heparins play an important role in cell growth, differentiation, migration and invasion. However, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. To determine the effect of heparin on gene expression, we performed a cDNA microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. In this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. We determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. We showed the importance of heparin mediated histone modifications and down-regulation of Enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxin-interacting protein. When we tested biological significance of these data; we observed that cells overexpressing thioredoxin-interacting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. Interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. In conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. This study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis.

  4. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    PubMed Central

    Stekel, Dov J; Jenkins, Dafyd J

    2008-01-01

    Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors

  5. Strong negative self regulation of prokaryotic transcription factors increases the intrinsic noise of protein expression.

    PubMed

    Stekel, Dov J; Jenkins, Dafyd J

    2008-01-18

    Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors.

  6. Sucrose increases calcium-dependent protein kinase and phosphatase activities in potato plants.

    PubMed

    Raíces, M; MacIntosh, G C; Ulloa, R M; Gargantini, P R; Vozza, N F; Téllez-Inón, M T

    2003-09-01

    The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.

  7. Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury.

    PubMed

    Jin, K; Li, W; Nagayama, T; He, X; Sinor, A D; Chang, J; Mao, X; Graham, S H; Simon, R P; Greenberg, D A

    2000-03-15

    T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

  8. Pancreatic protein hypersecretion and elevated plasma CCK: prerequisites for increased pancreatic growth?

    PubMed

    Rivard, N; Guan, D; Maouyo, D; Morisset, J

    1993-09-01

    This study was undertaken to establish if a correlation exists between chronic elevated pancreatic secretion and growth of the pancreas. Rats provided with jugular, pancreatic, biliary, duodenal, or ileal cannulas were fed throughout the experiment with a liquid diet continuously infused into the duodenum. Four days after surgery, control rats and those infused with cerulein (CE) 0.45 microgram/kg/h had their pancreatic juice returned into the duodenum. Two other groups had their pancreatic juice either totally diverted outside (DO) or returned into the ileum (DI). In all groups, bile was returned into the duodenum. Pancreatic juice was collected every 4 h for 4 days with volume and protein determined. After 4 days, rats were killed and their pancreata were evaluated for weight and contents of DNA, RNA, protein, amylase, and chymotrypsinogen. The average volumes/4 h were significantly increased by 259, 241, and 270% in DO, DI, and CE rats, respectively. Protein output remained at control levels in DO rats, whereas increases of 200 and 90% above control values were observed in DI and CE rats, respectively, during the last periods of collection. Constant drainage of pancreatic juice outside (DO) had no effect on pancreatic growth; on the contrary, its reinfusion into the ileum and constant cerulein infusion were associated with impressive growth of the pancreas, with cerulein being the most potent stimulus. In conclusion these data support the hypothesis that increased protein output is associated with pancreatic growth, a phenomenon mediated by endogenous cholecystokinin.

  9. Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

    PubMed Central

    Errington, Timothy M.; Macara, Ian G.

    2013-01-01

    The cellular response to DNA damage requires the coordination of many proteins involved in diverse molecular processes. Discrete molecular pathways are becoming increasingly well understood, but the interconnectivity and coordination of multiple pathways remains less clear. We now show that NCK, an adapter protein involved in cytoskeletal responses to tyrosine kinase receptor signaling, accumulates in the nucleus in response to DNA damage and this translocation can be blocked by specific inhibition of the ATR protein kinase. Strikingly, HeLa cells depleted of NCK undergo apoptosis shortly after UV irradiation, as monitored by caspase-3 cleavage and PARP cleavage. This rapid, hyperactive apoptosis in NCK depleted cells might be p53 dependent, because loss of NCK also increased UV-induced p53 phosphorylation. Importantly, depletion of SOCS7, which is necessary for NCK nuclear translocation, phenocopies NCK depletion, indicating the nuclear accumulation of NCK is responsible for these molecular events. There are two NCK isoforms that have mostly redundant functions, and although NCK2 appears to have a greater contribution, depletion of NCK1 or NCK2, led to increased p53 phosphorylation and early apoptosis after UV exposure. These data reveal a novel function for NCK in regulating p53 phosphorylation and apoptosis, and provide evidence for interconnectedness of growth factor signaling proteins and the DNA damage response. PMID:24086708

  10. Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2].

    PubMed

    Qiu, Quan-Sheng; Huber, Joan L; Booker, Fitzgerald L; Jain, Vanita; Leakey, Andrew D B; Fiscus, Edwin L; Yau, Peter M; Ort, Donald R; Huber, Steven C

    2008-08-01

    While exposure of C3 plants to elevated [CO2] would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the present study suggest that exposure of plants to elevated [CO2] can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated [CO2]. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated [CO2]. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated [CO2] or [O3] revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated [CO2].

  11. Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

    PubMed Central

    Grgurevic, Lovorka; Macek, Boris; Healy, David R.; Brault, Amy L.; Erjavec, Igor; Cipcic, Antonio; Grgurevic, Ivica; Rogic, Dunja; Galesic, Kresimir; Brkljacic, Jelena; Stern-Padovan, Ranka; Paralkar, Vishwas M.

    2011-01-01

    Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography–mass spectrometry, we identified BMP6, GDF15, and the BMP1–3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1–3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1–3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1–3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFβ1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1–3 increased the transcription of collagen type I, TGFβ1, β-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin β1 subunit. The profibrotic effect of BMP1–3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1–3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD. PMID:21415150

  12. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions.

    PubMed

    McReynolds, Jayme R; Donowho, Kyle; Abdi, Amin; McGaugh, James L; Roozendaal, Benno; McIntyre, Christa K

    2010-03-01

    Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a beta-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of beta-adrenoceptors with propranolol (0.5 microg/0.2 microL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA beta-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.

  13. Enhanced heterologous protein production in Pichia pastoris under increased air pressure.

    PubMed

    Lopes, Marlene; Oliveira, Carla; Domingues, Lucília; Mota, Manuel; Belo, Isabel

    2014-01-01

    Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. © 2014 American Institute of Chemical Engineers.

  14. Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses.

    PubMed

    Yang, L; Wang, B; Long, C; Wu, G; Zheng, H

    2007-11-23

    Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca(2+) channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca(2+) channel function.

  15. Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis

    PubMed Central

    Drummond, Micah J; Fry, Christopher S; Glynn, Erin L; Dreyer, Hans C; Dhanani, Shaheen; Timmerman, Kyle L; Volpi, Elena; Rasmussen, Blake B

    2009-01-01

    Muscle protein synthesis and mTORC1 signalling are concurrently stimulated following muscle contraction in humans. In an effort to determine whether mTORC1 signalling is essential for regulating muscle protein synthesis in humans, we treated subjects with a potent mTORC1 inhibitor (rapamycin) prior to performing a series of high-intensity muscle contractions. Here we show that rapamycin treatment blocks the early (1–2 h) acute contraction-induced increase (∼40%) in human muscle protein synthesis. In addition, several downstream components of the mTORC1 signalling pathway were also blunted or blocked by rapamycin. For instance, S6K1 phosphorylation (Thr421/Ser424) was increased post-exercise 6-fold in the control group while being unchanged with rapamycin treatment. Furthermore, eEF2 phosphorylation (Thr56) was reduced by ∼25% post-exercise in the control group but phosphorylation following rapamycin treatment was unaltered, indicating that translation elongation was inhibited. Rapamycin administration prior to exercise also reduced the ability of raptor to associate with mTORC1 during post-exercise recovery. Surprisingly, rapamycin treatment prior to resistance exercise completely blocked the contraction-induced increase in the phosphorylation of ERK1/2 (Thr202/Tyr204) and blunted the increase in MNK1 (Thr197/202) phosphorylation. However, the phosphorylation of a known target of MNK1, eIF4E (Ser208), was similar in both groups (P > 0.05) which is consistent with the notion that rapamycin does not directly inhibit MAPK signalling. We conclude that mTORC1 signalling is, in part, playing a key role in regulating the contraction-induced stimulation of muscle protein synthesis in humans, while dual activation of mTORC1 and ERK1/2 stimulation may be required for full stimulation of human skeletal muscle protein synthesis. PMID:19188252

  16. Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti

    PubMed Central

    2010-01-01

    Background The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell. Results Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport. Conclusion This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology. PMID:20573193

  17. Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men.

    PubMed

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K; Reitelseder, Søren; Drummond, Micah J; Schjerling, Peter; Scheike, Thomas; Serena, Anja; Holm, Lars

    2017-04-01

    The present study investigated whether well-tolerated light-load resistance exercise (LL-RE) affects skeletal muscle fractional synthetic rate (FSR) and anabolic intracellular signaling as a way to counteract age-related loss of muscle mass. Untrained healthy elderly (>65-yr-old) men were subjected to 13 h of supine rest. After 2.5 h of rest, unilateral LL-RE, consisting of leg extensions (10 sets, 36 repetitions) at 16% of 1 repetition maximum (RM), was conducted. Subsequently, the subjects were randomized to oral intake of 4 g of whey protein per hour (PULSE, n = 10), 28 g of whey protein at 0 h and 12 g of whey protein at 7 h postexercise (BOLUS, n = 10), or 4 g of maltodextrin per hour (placebo, n = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from the resting and the exercised leg of each subject. Myofibrillar FSR and activity of select targets from the mechanistic target of rapamycin complex 1-signaling cascade were analyzed from the biopsies. LL-RE increased myofibrillar FSR compared with the resting leg throughout the 10-h postexercise period. Phosphorylated (T308) AKT expression increased in the exercised leg immediately after exercise. This increase persisted in the placebo group only. Levels of phosphorylated (T37/46) eukaryotic translation initiation factor 4E-binding protein 1 increased throughout the postexercise period in the exercised leg in the placebo and BOLUS groups and peaked at 7 h. In all three groups, phosphorylated (T56) eukaryotic elongation factor 2 decreased in response to LL-RE. We conclude that resistance exercise at only 16% of 1 RM increased myofibrillar FSR, irrespective of nutrient type and feeding pattern, which indicates an anabolic effect of LL-RE in elderly individuals. This finding was supported by increased signaling for translation initiation and translation elongation in response to LL-RE.

  18. Low-dose gamma irradiation of food protein increases its allergenicity in a chronic oral challenge.

    PubMed

    Vaz, A F M; Souza, M P; Medeiros, P L; Melo, A M M A; Silva-Lucca, R A; Santana, L A; Oliva, M L V; Perez, K R; Cuccovia, I M; Correia, M T S

    2013-01-01

    Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.

  19. Increased activity of rat liver nucleolar protein kinase following triiodothyronine administration.

    PubMed

    Fugassa, E; Gallo, G; Pertica, M; Voci, A; Orunesu, M

    1977-12-08

    Triiodothyronine (T3) administration to thyroidectomized rats induces a significant increase in the nucleolus-associated protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. The general properties of the protein kinase solubilized from liver nucleoli have been investigated. Mg2+ (20 mM) is essential for the reaction and an appropriate concentration of NaCl (100 mM) is required to achieve maximal phosphorylation rates. The optimal pH for casein phosphorylation is 7.6. The kinase phosphorylates casein more efficiently than phosvitin and displays an almost undetectable activity towards histones and protamine. No significant stimulation of the kinase activity by cyclic AMP has been detected. The apparent Km values for casein and ATP are 1.5 mg/ml and 1.5-10(-5) M, respectively, and are not affected by the hormone administration.

  20. Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle.

    PubMed

    Petersen, Anne Marie Winther; Magkos, Faidon; Atherton, Philip; Selby, Anna; Smith, Kenneth; Rennie, Michael J; Pedersen, Bente Klarlund; Mittendorfer, Bettina

    2007-09-01

    Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.

  1. Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia*

    PubMed Central

    Jakubauskiene, Egle; Vilys, Laurynas; Makino, Yuichi; Poellinger, Lorenz; Kanopka, Arvydas

    2015-01-01

    The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles. PMID:26023237

  2. Mycobacterium tuberculosis increases IP-10 and MIG protein despite inhibition of IP-10 and MIG transcription.

    PubMed

    Bai, Xiyuan; Chmura, Kathryn; Ovrutsky, Alida R; Bowler, Russell P; Scheinman, Robert I; Oberley-Deegan, Rebecca E; Liu, Haiying; Shang, Shaobin; Ordway, Diane; Chan, Edward D

    2011-01-01

    Mycobacterium tuberculosis (MTB) has evolved methods to evade interferon-gamma (IFNγ) mediated protection. We sought to determine the effect of MTB infection on expression of IFNγ-inducible Protein 10 (IP-10) and Monokine Induced by IFNγ (MIG), two chemokines involved in host defense. MTB infection of THP-1 cells inhibited the transcription of IP-10 and MIG. A key mechanism for the inhibition is the disruption of binding of Signal Transduction and Activation of Transcription 1-alpha (STAT1α) to its cis-regulatory element, present in the 5'-flanking region of both IP-10 and MIG promoters. Use of inhibitors specific to the nuclear factor-kappa B (NFκB) and p38 mitogen-activated protein kinase (p38(mapk)) implicate these two signaling pathways in mediating the effect of MTB on the inhibition of IFNγ-induced IP-10 and MIG mRNA expression. Interestingly, despite transcriptional inhibition, there was an unexpected increase in IP-10 and MIG protein production after combined IFNγ and MTB stimulation. MTB also inhibited IFNγ induction of MIG mRNA but augmented MIG protein in primary human monocyte-derived macrophages. The synergy between MTB and IFNγ in the induction of IP-10 and MIG protein appears to involve novel post-transcriptional events that incorporates non-canonical functions of NFκB and p38(mapk). Published by Elsevier Ltd.

  3. Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling.

    PubMed

    Ohno, Yukihiro; Ishihara, Shizuka; Terada, Ryo; Kikuta, Miki; Sofue, Nobumasa; Kawai, Yoshiko; Serikawa, Tadao; Sasa, Masashi

    2009-12-18

    The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.

  4. Folding pathways of proteins with increasing degree of sequence identities but different structure and function.

    PubMed

    Giri, Rajanish; Morrone, Angela; Travaglini-Allocatelli, Carlo; Jemth, Per; Brunori, Maurizio; Gianni, Stefano

    2012-10-30

    Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G(A) (a 3-α helix fold) and G(B) (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G(A) domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G(B)s, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.

  5. Transdifferentiation of Bone Marrow Mesenchymal Stem Cells into the Islet-Like Cells: the Role of Extracellular Matrix Proteins.

    PubMed

    Pokrywczynska, Marta; Lewandowska, Marzena Anna; Krzyzanowska, Sandra; Jundzill, Arkadiusz; Rasmus, Marta; Warda, Karolina; Gagat, Maciej; Deptula, Aleksander; Helmin-Basa, Anna; Holysz, Marcin; Nowacki, Maciej; Buchholz, Lukasz; Bodnar, Magdalena; Marszalek, Andrzej; Grzanka, Alina; Jozwicki, Wojciech; Michalkiewicz, Jacek; Drewa, Tomasz

    2015-10-01

    Pancreatic islet implantation has been recently shown to be an efficient method of treatment for type 1 diabetes. However, limited availability of donor islets reduces its use. Bone morrow would provide potentially unlimited source of stem cells for generation of insulin-producing cells. This study was performed to evaluate the influence of extracellular matrix proteins like collagen, laminin, and vitronectin on bone marrow mesenchymal stem cells (BM-MSCs) transdifferentiation into islet-like cells (ILCs) in vitro. To our knowledge, this is the first report evaluating the importance of vitronectin in transdifferentiation of BM-MSCs into ILCs. Rat BM-MSCs were induced to ILCs using four-step protocol on plates coated with collagen type IV, laminin type I and vitronectin type I. Quantitative real-time PCR was performed to detect gene expression related to pancreatic β cell development. The induced cells expressed islet-related genes including: neurogenin 3, neurogenic differentiation 1, paired box 4, NK homeobox factor 6.1, glucagon, insulin 1 and insulin 2. Laminin but not collagen type IV or vitronectin enhanced expression of insulin and promoted formation of islet-like structures in monolayer culture. Laminin triggered transdifferentiation of BM-MSCs into ILCs.

  6. “Danger” Conditions Increase Sulfamethoxazole-Protein Adduct Formation in Human Antigen-Presenting Cells

    PubMed Central

    Lavergne, S. N.; Wang, H.; Callan, H. E.; Park, B. K.

    2009-01-01

    Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such “danger signals” on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 μM–2 mM; 5 min–24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1β, IL-6, IL-10; tumor necrosis factor-α; interferon-γ; and transforming growth factor-β], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H2O2), and hyperthermia (37.5–39.5°C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant to the

  7. "Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

    PubMed

    Lavergne, S N; Wang, H; Callan, H E; Park, B K; Naisbitt, D J

    2009-11-01

    Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant

  8. Hypocretin-1 causes G protein activation and increases ACh release in rat pons.

    PubMed

    Bernard, René; Lydic, Ralph; Baghdoyan, Helen A

    2003-10-01

    The effects of the arousal-promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague-Dawley rats. In vitro[35S]GTPgammaS autoradiography was used to test the hypothesis that hypocretin-1-stimulated G protein activation is concentration-dependent and blocked by the hypocretin receptor antagonist SB-334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration-response data revealed a significant (P < 0.001) effect of hypocretin-1 (2-2000 nm) in all brain regions examined. Maximal increases over control levels of [35S]GTPgammaS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB-334867 (2 micro m) significantly (P < 0.002) blocked hypocretin-1 (200 nm)-stimulated [35S]GTPgammaS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin-1 activates G proteins in arousal-related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin-1 increases ACh release in PnO. Dialysis delivery of hypocretin-1 (100 micro m) significantly (P < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.

  9. INCREASE IN ACTIVATED PROTEIN C MEDIATES ACUTE TRAUMATIC COAGULOPATHY IN MICE

    PubMed Central

    Chesebro, Brian B.; Rahn, Pamela; Carles, Michel; Esmon, Charles T.; Xu, Jun; Brohi, Karim; Frith, Daniel; Pittet, Jean-François; Cohen, Mitchell J.

    2013-01-01

    In severely injured and hypoperfused trauma patients, endogenous acute coagulopathy (EAC) is associated with an increased morbidity and mortality. Recent human data correlate this coagulopathy with activation of the protein C pathway. To examine the mechanistic role of protein C in the development of EAC, we used a mouse model of trauma and hemorrhagic shock, characterized by the combination of tissue injury and severe metabolic acidosis. Mice were subjected to one of four treatment groups: 1) C, control; 2) T, trauma (laparotomy); 3) H, hemorrhage (MAP, 35 mmHg × 60 min); 4) TH, trauma + hemorrhage. After 60 min, blood was drawn for analysis. Compared with C mice, the TH mice had a significantly elevated activated partial thromboplastin time (23.3 vs. 34.5 s) and significantly increased levels of activated protein C (aPC; 2.30 vs. 13.58 ng/mL). In contrast, T and H mice did not develop an elevated activated partial thromboplastin time or increased aPC. Selective inhibition of the anticoagulant property of aPC prevented the coagulopathy seen in response to trauma/hemorrhage (23.5 vs. 38.6 s [inhibitory vs. control monoclonal antibody]) with no impact on survival during the shock period. However, complete blockade of both the anticoagulant and cytoprotective functions of aPC caused 100% mortality within 45 min of shock, with histopathology evidence of pulmonary thrombosis and perivascular hemorrhage. These results indicate that our unique mouse model of T/H shock mimics our previous observations in trauma patients and demonstrates that EAC is mediated by the activation of the protein C pathway. In addition, the cytoprotective effect of protein C activation seems to be necessary for survival of the initial shock injury. PMID:19333141

  10. Increased C-reactive protein and fever in Japanese infants with food protein-induced enterocolitis syndrome.

    PubMed

    Kimura, Mitsuaki; Ito, Yasunori; Tokunaga, Fumika; Meguro, Takaaki; Shimomura, Masaki; Morishita, Hideaki; Seto, Shiro

    2016-09-01

    Increased C-reactive protein (CRP) and fever are observed in some infants with food protein-induced enterocolitis syndrome (FPIES) in Japan, but the reproducibility of these findings has not yet been confirmed on oral food challenge (OFC). Fourteen infants with FPIES induced by cow's milk (CM) formula were enrolled. OFC using CM formula was performed on each infant once or repeatedly (total 18 tests), with a stepwise incremental protocol in an infection-controlled setting. CRP was measured 24 h after the last ingestion of the CM formula. Increased CRP was observed in 11 of the 18 OFC conducted (median, 2.60 mg/dL; range, 0.22-4.84 mg/dL). Fever was induced in six occasions during OFC. Serum CRP in the patients with fever increased to median 3.76 mg/dL (range, <0.7-4.84 mg/dL), which was significantly higher than that of the patients without fever (median <0.1 mg/dL; range, <0.1-2.6 mg/dL; P < 0.001). CRP during OFC significantly correlated with that at disease onset (rs = 0.62, P < 0.02). Three of the four patients with fever at disease onset also had fever during OFC. Increased CRP and fever are reproducible during OFC in some infants with FPIES, suggesting that these are not accidental phenomena, but instead are associated with FPIES itself in Japanese patients. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

  11. Increased expression of the TIAR protein in the hippocampus of Alzheimer patients.

    PubMed

    Oleana, V H; Salehi, A; Swaab, D F

    1998-05-11

    T-cell restricted intracellular antigen related protein (TIAR) is an RNA-binding protein that is supposed to be involved in the process of stress-induced apoptosis. TIAR triggers DNA fragmentation in permeabilized thymocytes and its expression diminishes in the cell nucleus and rises simultaneously in the cytoplasm during Fas-induced cell death. Using a monoclonal antibody against TIAR, we stained different areas of the hippocampus from seven controls and 14 patients with Alzheimer's disease (AD). There was a clear expression of TIAR in the hippocampus of non-demented controls. Surprisingly, a significant increase was found in the expression of TIAR in the hippocampal area in AD. The increased expression of TIAR in AD may be related to the process of neurodegeneration in the hippocampus.

  12. A case of Pelizaeus-Merzbacher disease showing increased dosage of the proteolipid protein gene.

    PubMed

    Harding, B; Ellis, D; Malcolm, S

    1995-04-01

    Clinical, neuropathological and molecular genetic studies in a 9 month old boy with Pelizaeus-Merzbacher disease are described. The principal clinical features were developmental delay, nystagmus, stridor and seizures. Both brain and spinal cord showed almost complete absence of stainable central myelin, while cranial and spinal root myelin was preserved. Probes for cDNA in the boy and his asymptomatic mother indicated an increase in the dosage of proteolipid protein gene (of at least twofold) compared with controls.

  13. Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

    PubMed

    Ramachandran, V; Kostrubsky, V E; Komoroski, B J; Zhang, S; Dorko, K; Esplen, J E; Strom, S C; Venkataramanan, R

    1999-10-01

    Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.

  14. Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

    PubMed

    Adler, M J; Coronel, C; Shelton, E; Seegmiller, J E; Dewji, N N

    1991-01-01

    The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

  15. Redesigning Protein Cavities as a Strategy for Increasing Affinity in Protein-Protein Interaction: Interferon-γ Receptor 1 as a Model

    PubMed Central

    Biedermannová, Lada; Mikulecký, Pavel; Zahradník, Jiří; Charnavets, Tatsiana; Šebo, Peter

    2015-01-01

    Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity. PMID:26060819

  16. Nusinersen: antisense oligonucleotide to increase SMN protein production in spinal muscular atrophy.

    PubMed

    Paton, D M

    2017-06-01

    Patients with spinal muscular atrophy (SMA) have an autosomal recessive disease that limits their ability to produce survival motor neuron (SMN) protein in the CNS resulting in progressive wasting of voluntary muscles. Detailed studies over several years have demonstrated that phosphorothioate and 2'-O-methoxyethyl- modified antisense oligonucleotides (ASOs) targeting the ISS-N1 site increase SMN2 exon 7 inclusion, thus increasing levels of SMN protein in a dose- and time-dependent manner in liver, kidney and skeletal muscle, and CNS tissues only when administered intrathecally. On a dose basis, nusinersen was found to be the most potent ASO for SMN2 splicing correction in the CNS of adult mice. After nusinersen was found to increase levels of SMN protein in the CNS of mice and subhuman primates without causing significant adverse events, it was advanced into clinical studies in patients with SMA. These trials in SMA patients have demonstrated significant improvements in various measures of motor function and in progression to movement developments not normally seen in SMA patients. In addition, there have been significant extensions in life expectancy. These findings led to the U.S. and European approval of nusinersen for use in SMA patients of all ages. Copyright 2017 Clarivate Analytics.

  17. Increased protein and mRNA expression of resistin after dexamethasone administration.

    PubMed

    Sasayama, D; Hori, H; Nakamura, S; Yamamoto, N; Hattori, K; Teraishi, T; Ota, M; Kunugi, H

    2015-06-01

    Synthetic glucocorticoids such as dexamethasone are widely used to treat a variety of inflammatory and autoimmune conditions, but they may induce adverse events including hyperglycemia. To shed light on the effect and action mechanism of dexamethasone, we examined the alterations of gene expression levels caused by dexamethasone.Microarray analysis was performed on whole blood collected from 24 physically healthy subjects at baseline and after dexamethasone administration. The expression levels of resistin mRNA were found to be significantly increased after the dexamethasone administration. In a separate sample of 12 subjects, we examined plasma resistin protein levels and found that they were increased after dexamethasone administration. Furthermore, the plasma mRNA and protein levels of resistin were significantly higher in individuals who carried the A allele of RETN single nucleotide polymorphism rs3219175 than in those who did not carry the allele. There was no significant interaction between the genotype and dexamethasone administration. No significant correlation was found between plasma levels of cortisol and resistin. Consistent with previous studies, the genotype of RETN rs3219175 was a strong determinant of resistin levels. The present study showed that oral administration of dexamethasone increases the protein and mRNA levels of resistin irrespective of the rs3219175 genotype. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells.

  19. Increased thermal stability of proteins in the presence of amino acids.

    PubMed Central

    Taneja, S; Ahmad, F

    1994-01-01

    This study is a systematic attempt to understand the roles of osmolytes in protecting proteins against denaturing stress. Thermal denaturation of cytochrome c has been studied in the presence of various concentrations of all L-amino acids that are more hydrophobic than glycine and have a solubility of 0.1 M or higher in water at 25 degrees C. The basic observations are as follows. (1) Arginine and histidine destabilize the native protein; both Tm (the midpoint of thermal transition) and delta GDH2O (25 degrees C) (the Gibbs energy of stabilization) decrease with increasing amino acid concentration. (2) Isoleucine, leucine and phenylalanine have no effect on Tm and deltaGDH2O (25 degrees C). (3) Valine and less hydrophobic amino acids stabilize the protein in terms of Tm but deltaGDH2O (25 degrees C) is unchanged. This observation was confirmed by the study of isothermal denaturation of cytochrome c by guanidinium chloride which suggested that delta GDH2O is independent of osmolyte concentration, but Cm (the midpoint of transition) is increased in their presence. (4) In the case of stabilizers, change in Tm/mol of amino acid decreases with increasing hydrophobicity of these osmolytes. PMID:7945233

  20. Geranylgeranylacetone attenuates hepatic fibrosis by increasing the expression of heat shock protein 70

    PubMed Central

    HE, WEI; ZHUANG, YUN; WANG, LIANGZHI; QI, LEI; CHEN, BINFANG; WANG, MEI; SHAO, DONG; CHEN, JIANPING

    2015-01-01

    Increasing evidence has demonstrated that the heat shock protein 70 (HSP70) gene may be closely associated with tissue fibrosis; however, the association between HSP70 and liver fibrosis remains to be fully elucidated. The present study hypothesized that geranylgeranylacetone (GGA) exerts beneficial effects on liver fibrosis though upregulation of the expression of HSP70. Liver fibrosis was induced in rats using carbon tetrachloride (CCl4). The rats were subsequently divided into three groups: Control group, CCl4 model group and CCl4 model + GGA group. Liver fibrosis in the rats was evaluated using hematoxylin and eosin staining, Masson's trichrome staining and Sirius red staining. The levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin were determined using an automated biochemistry analyzer. The levels of total hepatic hydroxyproline were also determined. The expression levels of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) were determined using immunofluorescence staining and western blotting, and the protein expression levels of HSP70 were determined using western blotting. The CCl4-induced rats exhibited liver fibrosis, increased hydroxyproline content, impaired liver function, upregulated expression levels of the α-SMA and TGF-β1 pro-fibrogenic proteins, and increased expression of HSP70, compared with the control group. These changes were attenuated by treatment with GGA. These results demonstrated that GGA exerted beneficial effects in CCl4-induced liver fibrosis via upregulating the expression of HSP70. PMID:26165998

  1. Geranylgeranylacetone attenuates hepatic fibrosis by increasing the expression of heat shock protein 70.

    PubMed

    He, Wei; Zhuang, Yun; Wang, Liangzhi; Qi, Lei; Chen, Binfang; Wang, Mei; Shao, Dong; Chen, Jianping

    2015-10-01

    Increasing evidence has demonstrated that the heat shock protein 70 (HSP70) gene may be closely associated with tissue fibrosis; however, the association between HSP70 and liver fibrosis remains to be fully elucidated. The present study hypothesized that geranylgeranylacetone (GGA) exerts beneficial effects on liver fibrosis though upregulation of the expression of HSP70. Liver fibrosis was induced in rats using carbon tetrachloride (CCl4). The rats were subsequently divided into three groups: Control group, CCl4 model group and CCl4 model + GGA group. Liver fibrosis in the rats was evaluated using hematoxylin and eosin staining, Masson's trichrome staining and Sirius red staining. The levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin were determined using an automated biochemistry analyzer. The levels of total hepatic hydroxyproline were also determined. The expression levels of α‑smooth muscle actin (α‑SMA) and transforming growth factor‑β1 (TGF‑β1) were determined using immunofluorescence staining and western blotting, and the protein expression levels of HSP70 were determined using western blotting. The CCl4‑induced rats exhibited liver fibrosis, increased hydroxyproline content, impaired liver function, upregulated expression levels of the α‑SMA and TGF‑β1 pro‑fibrogenic proteins, and increased expression of HSP70, compared with the control group. These changes were attenuated by treatment with GGA. These results demonstrated that GGA exerted beneficial effects in CCl4‑induced liver fibrosis via upregulating the expression of HSP70.

  2. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex.

    PubMed Central

    Shlaes, D M; Shlaes, J H; Vincent, S; Etter, L; Fey, P D; Goering, R V

    1993-01-01

    In the recent clinical trials of teicoplanin therapy of endocarditis caused by Staphylococcus aureus, at least one instance of the emergence of teicoplanin-resistant strains during therapy has been reported (G.W. Kaatz, S. M. Seo, N. J. Dorman, and S. A. Lerner, J. Infect. Dis 162:103-108, 1990). We have confirmed, using conventional electrophoresis of EcoRI-digested chromosomal DNA and pulsed-field gel electrophoresis of SmaI-digested chromosomal DNA, that the resistant strain (12873) (MIC, 16 micrograms/ml) is genetically very similar to the susceptible parent (12871) (MIC, 4 micrograms/ml). Kaatz et al. were able to select spontaneous teicoplanin-resistant mutants (10(-9)), suggesting that a single gene might be involved. We have shown that the mutation is highly stable during growth in the absence of teicoplanin. Using Tn551, we have selected insertion mutants of 12873 that become teicoplanin susceptible. We have examined a number of aspects of cell wall physiology in strains 12871 and 12873 and the teicoplanin-susceptible Tn551 mutants of 12873. 12873 was more susceptible to lysostaphin lysis than 12871 and the susceptible Tn551 derivatives of 12873. Autolysis in phosphate buffer (pH 7.5) and cell wall turnover rates were similar in 12871 and 12873. An analysis of membrane proteins revealed the expression of a ca. 35-kDa protein and increased expression of both polypeptides of penicillin-binding protein (PBP) 2 (PBP2) in 12873 relative to 12871 and the Tn551 mutants of 12873. This increased expression was not related to PBP2', since both strains were susceptible to oxacillin in 2% NaCl (MIC, < or = 0.25 microgram/ml) and cellular DNA from neither strain hybridized with a specific mec gene probe. Two independent Tn551 inserts have been mapped to a ca. 117-kb SmaI fragment of the chromosome. These data suggest the possibility that the mutation resulting in resistance to teicoplanin involves the regulation of expression of both polypeptides of PBP2 and a 35-k

  3. Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

    PubMed

    Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

  4. Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

    PubMed Central

    Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

  5. Inadequate Dietary Protein Increases Hunger and Desire to Eat in Younger and Older Men12

    PubMed Central

    Apolzan, John W.; Carnell, Nadine S.; Mattes, Richard D.; Campbell, Wayne W.

    2008-01-01

    Many older people experience changes in appetite and consume marginal or inadequate dietary protein. This study was designed to examine the appetitive responses to habitual protein intakes that span the range of adequacy in younger and older men. Twenty-two men (12 younger, aged 21–43 y and 10 older, aged 63–79 y) completed, in random order, three 18-d trials that involved consumption of individualized, isoenergetic menus providing 1.00, 0.75, and 0.50 g protein · kg BW−1 · d−1, which were 125% (trial P125), 94% (trial P94), and 63% (trial P63) of the Recommended Dietary Allowance for protein. Near the end of each trial, the subjects recorded appetitive sensations hourly throughout one day using a visual analogue scale. Independent of age, ratings of hunger were lower for P125 (1.3 ± 0.5 cm) than P94 (1.8 ± 0.8 cm) and P63 (1.8 ± 0.6 cm) (P = 0.037), and desire to eat was lower during the P125 trial (1.4 ± 0.5 cm), compared with the P63 trial (2.1 ± 0.7 cm) (P = 0.003), and P94(1.8 ± 0.7 cm) was not different when compared with P63 and P125. Protein intake did not influence fullness. These results show that younger and older men who consume inadequate protein experience appetite changes that may promote increased food intake. PMID:17513410

  6. Unraveling protein folding mechanism by analyzing the hierarchy of models with increasing level of detail

    NASA Astrophysics Data System (ADS)

    Hayashi, Tomohiko; Yasuda, Satoshi; Škrbić, Tatjana; Giacometti, Achille; Kinoshita, Masahiro

    2017-09-01

    Taking protein G with 56 residues for a case study, we investigate the mechanism of protein folding. In addition to its native structure possessing α-helix and β-sheet contents of 27% and 39%, respectively, we construct a number of misfolded decoys with a wide variety of α-helix and β-sheet contents. We then consider a hierarchy of 8 different models with increasing level of detail in terms of the number of entropic and energetic physical factors incorporated. The polyatomic structure is always taken into account, but the side chains are removed in half of the models. The solvent is formed by either neutral hard spheres or water molecules. Protein intramolecular hydrogen bonds (H-bonds) and protein-solvent H-bonds (the latter is present only in water) are accounted for or not, depending on the model considered. We then apply a physics-based free-energy function (FEF) corresponding to each model and investigate which structures are most stabilized. This special approach taken on a step-by-step basis enables us to clarify the role of each physical factor in contributing to the structural stability and separately elucidate its effect. Depending on the model employed, significantly different structures such as very compact configurations with no secondary structures and configurations of associated α-helices are optimally stabilized. The native structure can be identified as that with lowest FEF only when the most detailed model is employed. This result is significant for at least the two reasons: The most detailed model considered here is able to capture the fundamental aspects of protein folding notwithstanding its simplicity; and it is shown that the native structure is stabilized by a complex interplay of minimal multiple factors that must be all included in the description. In the absence of even a single of these factors, the protein is likely to be driven towards a different, more stable state.

  7. Neuromuscular electrical stimulation increases muscle protein synthesis in elderly type 2 diabetic men.

    PubMed

    Wall, Benjamin T; Dirks, Marlou L; Verdijk, Lex B; Snijders, Tim; Hansen, Dominique; Vranckx, Pascal; Burd, Nicholas A; Dendale, Paul; van Loon, Luc J C

    2012-09-01

    Physical activity is required to attenuate the loss of skeletal muscle mass with aging. Short periods of muscle disuse, due to sickness or hospitalization, reduce muscle protein synthesis rates, resulting in rapid muscle loss. The present study investigates the capacity of neuromuscular electrical stimulation (NMES) to increase in vivo skeletal muscle protein synthesis rates in older type 2 diabetes patients. Six elderly type 2 diabetic men (70 ± 2 yr) were subjected to 60 min of one-legged NMES. Continuous infusions with L-[ring-¹³C₆]phenylalanine were applied, with blood and muscle samples being collected regularly to assess muscle protein synthesis rates in both the stimulated (STIM) and nonstimulated control (CON) leg during 4 h of recovery after NMES. Furthermore, mRNA expression of key genes implicated in the regulation of muscle mass were measured over time in the STIM and CON leg. Muscle protein synthesis rates were greater in the STIM compared with the CON leg during recovery from NMES (0.057 ± 0.008 vs. 0.045 ± 0.008%/h, respectively, P < 0.01). Skeletal muscle myostatin mRNA expression in the STIM leg tended to increase immediately following NMES compared with the CON leg (1.63- vs. 1.00-fold, respectively, P = 0.07) but strongly declined after 2 and 4 h of recovery in the STIM leg only. In conclusion, this is the first study to show that NMES directly stimulates skeletal muscle protein synthesis rates in vivo in humans. NMES likely represents an effective interventional strategy to attenuate muscle loss in elderly individuals during bed rest and/or in other disuse states.

  8. Heat Shock Protein translocation induced by membrane fluidization increases tumor-cell sensitivity to chemotherapeutic drugs.

    PubMed

    Dempsey, Nina C; Ireland, H Elyse; Smith, Carly M; Hoyle, Christine F; Williams, John H H

    2010-10-28

    Treatment of chronic lymphocytic leukemia (CLL) remains a challenge due to the frequency of drug resistance amongst patients. Improving the delivery of chemotherapeutic agents while reducing the expression of anti-apoptotic Heat Shock Proteins (HSPs) within the cancer cells may facilitate in overcoming this drug resistance. We demonstrate for the first time that sub-lethal doses of chemotherapeutic agents can be combined with membrane fluidizing treatments to produce a significant increase in drug efficacy and apoptosis in vitro. We show that fluidizers result in a transient decrease in intracellular HSPs, resulting in increased tumor-cell sensitivity and a membrane-associated induction of HSP gene expression.

  9. Increased signaling entropy in cancer requires the scale-free property of protein interaction networks

    PubMed Central

    Teschendorff, Andrew E.; Banerji, Christopher R. S.; Severini, Simone; Kuehn, Reimer; Sollich, Peter

    2015-01-01

    One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology. PMID:25919796

  10. Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity.

    PubMed

    McCarty, M F

    1999-12-01

    Amino acids modulate the secretion of both insulin and glucagon; the composition of dietary protein therefore has the potential to influence the balance of glucagon and insulin activity. Soy protein, as well as many other vegan proteins, are higher in non-essential amino acids than most animal-derived food proteins, and as a result should preferentially favor glucagon production. Acting on hepatocytes, glucagon promotes (and insulin inhibits) cAMP-dependent mechanisms that down-regulate lipogenic enzymes and cholesterol synthesis, while up-regulating hepatic LDL receptors and production of the IGF-I antagonist IGFBP-1. The insulin-sensitizing properties of many vegan diets--high in fiber, low in saturated fat--should amplify these effects by down-regulating insulin secretion. Additionally, the relatively low essential amino acid content of some vegan diets may decrease hepatic IGF-I synthesis. Thus, diets featuring vegan proteins can be expected to lower elevated serum lipid levels, promote weight loss, and decrease circulating IGF-I activity. The latter effect should impede cancer induction (as is seen in animal studies with soy protein), lessen neutrophil-mediated inflammatory damage, and slow growth and maturation in children. In fact, vegans tend to have low serum lipids, lean physiques, shorter stature, later puberty, and decreased risk for certain prominent 'Western' cancers; a vegan diet has documented clinical efficacy in rheumatoid arthritis. Low-fat vegan diets may be especially protective in regard to cancers linked to insulin resistance--namely, breast and colon cancer--as well as prostate cancer; conversely, the high IGF-I activity associated with heavy ingestion of animal products may be largely responsible for the epidemic of 'Western' cancers in wealthy societies. Increased phytochemical intake is also likely to contribute to the reduction of cancer risk in vegans. Regression of coronary stenoses has been documented during low-fat vegan diets

  11. Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women.

    PubMed

    Uenishi, K; Ishida, H; Toba, Y; Aoe, S; Itabashi, A; Takada, Y

    2007-03-01

    Effect of milk basic protein on bone metabolism in healthy young women. Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone mineral density and bone metabolism of healthy young women. Thirty-five healthy young women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indexes of bone metabolism were measured at 0, 3 and 6 months. All subjects completed the study in accordance with the protocol. The mean rate of gain of lumbar BMD in the MBP group (1.57%) was significantly higher than in the placebo group (0.13%, P=0.042). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased, and serum osteocalcin was significantly increased in the MBP group at 6 months. These results suggested that MBP supplementation was effective in increasing BMD in young women and that this increase in BMD may be primarily mediated through the promotion of bone formation and inhibition of bone resorption by MBP supplementation.

  12. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    PubMed

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  13. ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

    PubMed

    Muñoz-Félix, José M; Perretta-Tejedor, Nuria; Eleno, Nélida; López-Novoa, José M; Martínez-Salgado, Carlos

    2014-06-01

    Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

  14. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  15. Oral N-carbamylglutamate supplementation increases protein synthesis in skeletal muscle of piglets.

    PubMed

    Frank, Jason W; Escobar, Jeffery; Nguyen, Hanh V; Jobgen, Scott C; Jobgen, Wenjuan S; Davis, Teresa A; Wu, Guoyao

    2007-02-01

    This study investigated the potential mechanisms by which oral supplementation of N-carbamylglutamate (NCG), an analogue of endogenous N-acetylglutamate (an activator of arginine synthesis) increases growth rate in sow-reared piglets. Two piglets of equal body weight (BW) and of the same gender from each lactating sow were allotted to receive oral administration of 0 (control) or 50 mg of NCG/kg BW every 12 h for 7 d. Piglets (n=32; BW=3 kg) were studied in the food-deprived or fed state following the 7 d of treatment. Overnight food-deprived piglets were given NCG or water (control) at time 0 and 60 min. Piglets studied in the fed state were gavage-fed sow's milk with their respective NCG treatment at 0 and 60 min. At 60 min, the piglets were administered a flooding dose of [3H]phenylalanine and killed at 90 min to measure tissue protein synthesis. Piglets treated with NCG gained 28% more weight than control pigs (P<0.001) over the 7-d period. Fed pigs had greater rates of protein synthesis in longissimus dorsi and gastrocnemius muscles and duodenum compared with food-deprived pigs (P<0.001). Absolute protein synthesis rates in longissimus dorsi (P=0.050) and gastrocnemius (P=0.068) muscles were 30 and 21% greater, respectively, in NCG-treated compared with control pigs. Piglets supplemented with NCG also had greater plasma concentrations of arginine and somatotropin than control pigs (P<0.001). The results suggest that oral NCG supplementation increases plasma arginine and somatotropin levels, leading to an increase in growth rate and muscle protein synthesis in nursing piglets.

  16. C-reactive protein increases plasminogen activator inhibitor–1 expression in human endothelial cells

    PubMed Central

    Chen, Changyi; Nan, Bicheng; Lin, Peter; Yao, Qizhi

    2010-01-01

    C-reactive protein (CRP) is an inflammatory marker which predicts cardiovascular disease. However, it is not fully understood whether CRP has direct effects on endothelial functions and gene expression. The purpose of current study was to determine the effects and molecular mechanisms of CRP on the expression of plasminogen activator inhibitor-1 (PAI-1) in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations for different durations. PAI-1 mRNA, protein and enzyme activities were studied. The effects of CRP on MAPK p38 phosphorylation was also studied by Bio-Plex luminex immunoassay. In addition, other types of human endothelial cells isolated from umbilical vein, skin, and lung microvessels were tested. CRP significantly increased PAI-1 mRNA levels in a time- and concentration-dependent manner. The protein level and enzyme activity of PAI-1 in the supernatant of CRP-treated HCAEC cultures were significantly increased. Anti-CD32 antibody effectively blocked CRP-induced PAI-1 mRNA expression. In addition, CRP significantly increased CD32 mRNA levels and enhanced phosphorylation of MAPK p38. Furthermore, antioxidant curcumin dramatically inhibited CRP-induced PAI-1 mRNA expression. The effect of CRP on PAI-1 expression was also confirmed in other types of human endothelial cells. In conclusion, CRP significantly increased the expression of PAI-1 in HCAEC and other human endothelial cells. CRP also increased its receptor CD32 expression which may further enhance its action. CRP-induced PAI-1 expression may be mediated by oxidative stress and p38 signal pathway as antioxidant effectively blocks the effect of CRP on HCAEC. PMID:17949793

  17. Biomarker-calibrated Energy and Protein Consumption and Increased Cancer Risk Among Postmenopausal Women

    PubMed Central

    Shaw, Pamela A.; Bingham, Sheila A.; Beresford, Shirley A. A.; Caan, Bette; Neuhouser, Marian L.; Patterson, Ruth E.; Stefanick, Marcia L.; Satterfield, Suzanne; Thomson, Cynthia A.; Snetselaar, Linda; Thomas, Asha; Tinker, Lesley F.

    2009-01-01

    The authors previously reported equations, derived from the Nutrient Biomarker Study within the Women's Health Initiative, that produce calibrated estimates of energy, protein, and percentage of energy from protein consumption from corresponding food frequency questionnaire estimates and data on other factors, such as body mass index, age, and ethnicity. Here, these equations were applied to yield calibrated consumption estimates for 21,711 women enrolled in the Women's Health Initiative dietary modification trial comparison group and 59,105 women enrolled in the observational study. These estimates were related prospectively to total and site-specific invasive cancer incidence (1993–2005). In combined cohort analyses that do not control for body mass, uncalibrated energy was not associated with total cancer incidence or site-specific cancer incidence for most sites, whereas biomarker-calibrated energy was positively associated with total cancer (hazard ratio = 1.18, 95% confidence interval: 1.10, 1.27, for 20% consumption increase), as well as with breast, colon, endometrial, and kidney cancer (respective hazard ratios of 1.24, 1.35, 1.83, and 1.47). Calibrated protein was weakly associated, and calibrated percentage of energy from protein was inversely associated, with total cancer. Calibrated energy and body mass index associations were highly interdependent. Implications for the interpretation of nutritional epidemiology studies are described. PMID:19258487

  18. Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

    PubMed Central

    Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

    2016-01-01

    ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

  19. The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

    PubMed Central

    Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

    2009-01-01

    Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

  20. Soy protein diet increases skilled forelimb reaching function after stroke in rats.

    PubMed

    Cheatwood, Joseph L; Burnet, Derek; Butteiger, Dustie N; Banz, William J

    2011-01-20

    Stroke is a leading cause of lasting disability. Dietary strategies aimed at increasing post-stroke outcomes are lifestyle alterations which could be easily implemented by people at risk of occlusive stroke. Soy diets have been demonstrated to provide some benefits in the short term following stroke, but longer time periods have not been studied. Further, carefully defined diets containing soy protein isolates have not been investigated. In the current study, male Long Evans Hooded rats were fed semi-purified diets containing either sodium caseinate or soy protein isolate. Rats were trained to perform the skilled forelimb reaching task and subsequently underwent unilateral middle cerebral artery occlusion (MCAO) to induce a stroke lesion. After stroke, rats remained on the same diet and were tested daily for a period of 8 weeks to observe their performance on the skilled forelimb reaching task. In the first week following stroke, rats receiving the soy protein-containing diet (SP) demonstrated less severe reaching deficits than rats fed the Na caseinate-containing diet (CAS) (p<0.05). These results suggest that a soy protein-based diet provides significant protection from neurological damage following MCAO stroke in rats.

  1. Elevated maternal C-reactive protein and increased risk of schizophrenia in a national birth cohort.

    PubMed

    Canetta, Sarah; Sourander, Andre; Surcel, Heljä-Marja; Hinkka-Yli-Salomäki, Susanna; Leiviskä, Jaana; Kellendonk, Christoph; McKeague, Ian W; Brown, Alan S

    2014-09-01

    The objective of the present study was to investigate an association between early gestational C-reactive protein, an established inflammatory biomarker, prospectively assayed in maternal sera, and schizophrenia in a large, national birth cohort with an extensive serum biobank. A nested case-control design from the Finnish Prenatal Study of Schizophrenia cohort was utilized. A total of 777 schizophrenia cases (schizophrenia, N=630; schizoaffective disorder, N=147) with maternal sera available for C-reactive protein testing were identified and matched to 777 control subjects in the analysis. Maternal C-reactive protein levels were assessed using a latex immunoassay from archived maternal serum specimens. Increasing maternal C-reactive protein levels, classified as a continuous variable, were significantly associated with schizophrenia in offspring (adjusted odds ratio=1.31, 95% confidence interval=1.10-1.56). This finding remained significant after adjusting for potential confounders, including maternal and parental history of psychiatric disorders, twin/singleton birth, urbanicity, province of birth, and maternal socioeconomic status. This finding provides the most robust evidence to date that maternal inflammation may play a significant role in schizophrenia, with possible implications for identifying preventive strategies and pathogenic mechanisms in schizophrenia and other neurodevelopmental disorders.

  2. Staining proteins: a simple method to increase the sensitivity of ellipsometric measurements in adsorption studies.

    PubMed

    Nejadnik, M Reza; Garcia, Carlos D

    2011-01-01

    This communication describes a simple way to improve the sensitivity of spectroscopic ellipsometry, when applied to monitor the adsorption of proteins to solid surfaces. The method described herein is based on the reaction of a commercially available dye (Coomassie brilliant blue G) with the adsorbed proteins and the subsequent analysis by spectroscopic ellipsometry. In order to demonstrate the potential advantages of this method, the adsorption of bovine serum albumin to an antifouling coating was also investigated. According to our results, the modification with the dye significantly affects the optical properties of the adsorbed protein layer, which can be represented using a simple optical model (Lorentz). In general, the proposed modification increases the sensitivity of the detection by 2.5 ± 0.4-fold and enables the analysis of thin layers of adsorbed protein not obtainable by conventional methods. These results particularly reveal the importance of the proposed modification for the evaluation of low adsorbing substrates and antifouling coatings. Published by Elsevier B.V.

  3. Structural requirements for intracellular processing and sorting of bactericidal/permeability-increasing protein (BPI): comparison with lipopolysaccharide-binding protein.

    PubMed

    Bülow, E; Gullberg, U; Olsson, I

    2000-11-01

    The bactericidal/permeability-increasing protein (BPI), which is stored in the azurophil granules of neutrophils, and the circulating lipopolysaccharide-binding protein (LBP) share the same structure. Both bind lipopolysaccharide of gram-negative bacteria through their amino-terminal domains. The carboxy-terminal domain of BPI promotes bacterial attachment to phagocytes, whereas the corresponding domain of LBP delivers lipopolysaccharide to monocytes/macrophages. Our aim was to investigate the role of the amino-and carboxy-terminal domains of BPI and LBP for sorting and storage in myeloid cells after transfection of cDNA to two rodent hematopoietic cell lines. Full-length BPI and LBP were both targeted for storage in these cells. Deletion of the carboxy-terminal half of BPI resulted in storage followed by degradation while the reciprocal deletion of the amino-terminal half led to retention in the endoplasmic reticulum for proteasomal degradation. Chimeras between halves of BPI and LBP were also targeted for storage, but those containing carboxy-terminal BPI had the highest stability, again indicating a role for the carboxy-terminal domain of BPI in protection against degradation. Therefore, we propose a critical stability function for the hydrophobic carboxy-terminal domain of BPI during intracellular sorting for storage while the amino-terminal domain may confer targeting for storage.

  4. The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

    PubMed Central

    Pinkenburg, Olaf; Meyer, Torben; Bannert, Norbert; Norley, Steven; Bolte, Kathrin; Czudai-Matwich, Volker; Herold, Susanne; Gessner, André; Schnare, Markus

    2016-01-01

    In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely. PMID:27273104

  5. In vivo protein targets for increased quinoprotein adduct formation in aged substantia nigra.

    PubMed

    Yu, Guohua; Liu, Huiyan; Zhou, Wei; Zhu, Xuewei; Yu, Chao; Wang, Na; Zhang, Yi; Ma, Ji; Zhao, Yulan; Xu, Yuanyuan; Liao, Lujian; Ji, Hongfang; Yuan, Chonggang; Ma, Jiyan

    2015-09-01

    The selective vulnerability of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease, a late age onset neurodegenerative disorder, indicates the involvement of dopamine metabolism in the pathogenesis. Dopamine oxidation produces dopamine o-quinone, which covalently modifies cysteinyl proteins forming quinoprotein adduct. Although quinoprotein formation correlates with increased dopaminergic neurotoxicity, the in vivo protein targets for quinone modification remain unclear. Using two-dimensional gel electrophoresis and nitroblue tetrazolium/glycinate redox-cycling staining, we compared quinoprotein adducts in the substantia nigra of 2- and 15-month old rats and for the first time identified the in vivo protein targets with increased quinone modification in aged substantia nigra. Interestingly, several key enzymes in energy metabolism and mitochondrial function were selectively modified by quinone during aging. In vitro analyses confirmed that two of identified enzymes, l-lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were readily conjugated by dopamine o-quinone, resulting in a significant reduction in enzyme activity. Since the proteomic approach to detect quinoprotein adducts represents a single analysis comparing pools of substantia nigra from young or old rats, these findings need to be verified in the future. Nonetheless, our results reveal that the enzymatic activity of LDH and MDH can be compromised by quinone modification, suggesting a role of energy metabolism impairment in the selective vulnerability of aged substantia nigra dopaminergic neurons in Parkinson's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex.

    PubMed

    Wichern, Franziska; Jensen, Majbrit M; Christensen, Ditte Z; Mikkelsen, Jens D; Gondré-Lewis, Marjorie C; Thomsen, Morten S

    2017-03-27

    The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater degree in the frontal cortex (FC) compared with the hippocampus (HIP). We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the FC at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during early (PND8-14) or late (PND54-60) postnatal stages did not affect NACHO protein levels in the FC or HIP, neither did exposure to high doses of the selective α7 nAChR agonists SSR180711, A-582941, or PNU-282987. However, we found significantly increased NACHO protein levels in the FC of PND36 rats after a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal development. The effect of PNU-120596 further suggests that the increase in NACHO expression is caused by activation rather than desensitization of nAChRs. Copyright © 2017 IBRO. All rights reserved.

  7. Amyloid-beta oligomers increase the localization of prion protein at the cell surface.

    PubMed

    Caetano, Fabiana A; Beraldo, Flavio H; Hajj, Glaucia N M; Guimaraes, Andre L; Jürgensen, Sofia; Wasilewska-Sampaio, Ana Paula; Hirata, Pedro H F; Souza, Ivana; Machado, Cleiton F; Wong, Daisy Y-L; De Felice, Fernanda G; Ferreira, Sergio T; Prado, Vania F; Rylett, R Jane; Martins, Vilma R; Prado, Marco A M

    2011-05-01

    In Alzheimer's disease, the amyloid-β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aβ oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aβ oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.

  8. Chronic enteral leucine supplementation of a low protein diet increases skeletal muscle protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation

    USDA-ARS?s Scientific Manuscript database

    Leucine appears to be the key amino acid that positively regulates mTOR signalling. We hypothesized that prolonged feeding (24 hours) of a Leu supplemented low protein (LP) diet in neonatal pigs will increase protein synthesis in skeletal muscle to a rate similar to that of a high protein diet (HP)....

  9. AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization

    PubMed Central

    Rada, Cristina; Jarvis, John M.; Milstein, César

    2002-01-01

    Somatic hypermutation generates variants of antibody genes and underpins the affinity maturation of antibodies. It is restricted to the V-gene segments, and although it decays exponentially toward the 3′end, it includes recognizable hot spots. Although the detailed mechanism of hypermutation remains elusive, the process may take place in two separate stages, preferentially targeting G/Cs in the first and A/Ts in the second stage. It seems that MSH2 is involved in the second stage, and that activation induced deaminase (AID) is implicated in the control of hypermutation. The constitutively hypermutating cell line Ramos expresses AID, and we have prepared transfectants that express a chimeric AID-green fluorescent protein. The fluorescence is strongly detected in the cytoplasm but not in the nucleus. Yet, the chimeric protein increases the hypermutation rate either directly or, more likely, indirectly, by favoring the transport of AID into the nucleus. Thus, in Ramos, AID seems to be rate limiting. Unexpectedly, the proportion of deletions also is increased. The increase in mutation rate detected by a fast cytofluorimetric method based on the accumulation of sIgM-loss mutants correlates with the increase measured by mutations defined by sequence analysis. The higher mutation rate is largely explained by the higher proportion of mutated clones, indicating that AID controls the number of cells that undergo hypermutation but not the number of mutations that are incorporated in each mutation round. PMID:12011459

  10. Computational evidence that fast translation speed can increase the probability of cotranslational protein folding.

    PubMed

    Wang, Ercheng; Wang, Jun; Chen, Changjun; Xiao, Yi

    2015-10-21

    Translation speed can affect the cotranslational folding of nascent peptide. Experimental observations have indicated that slowing down translation rates of codons can increase the probability of protein cotranslational folding. Recently, a kinetic modeling indicates that fast translation can also increase the probability of cotranslational protein folding by avoiding misfolded intermediates. We show that the villin headpiece subdomain HP35 is an ideal model to demonstrate this phenomenon. We studied cotranslational folding of HP35 with different fast translation speeds by all-atom molecular dynamics simulations and found that HP35 can fold along a well-defined pathway that passes the on-pathway intermediate but avoids the misfolded off-pathway intermediate in certain case. This greatly increases the probability of HP35 cotranslational folding and the approximate mean first passage time of folding into native state is about 1.67μs. Since we also considered the space-confined effect of the ribosomal exit tunnel on the cotranslational folding, our simulation results suggested alternative mechanism for the increasing of cotranslational folding probability by fast translation speed.

  11. Uncoupling Protein 2 Increases Susceptibility to Lipopolysaccharide-Induced Acute Lung Injury in Mice

    PubMed Central

    Wang, Qin; Wang, Jianchun; Hu, Mingdong; Yang, Yu; Guo, Liang; Xu, Jing; Lei, Chuanjiang; Jiao, Yan; Xu, JianCheng

    2016-01-01

    Uncoupling protein 2 (UCP2) is upregulated in patients with systemic inflammation and infection, but its functional role is unclear. We up- or downregulated UCP2 expression using UCP2 recombinant adenovirus or the UCP2 inhibitor, genipin, in lungs of mice, and investigated the mechanisms of UCP2 in ALI. UCP2 overexpression in mouse lungs increased LPS-induced pathological changes, lung permeability, lung inflammation, and lowered survival rates. Furthermore, ATP levels and mitochondrial membrane potential were decreased, while reactive oxygen species production was increased. Additionally, mitogen-activated protein kinases (MAPKs) activity was elevated, which increased the sensitivity to LPS-induced apoptosis and inflammation. LPS-induced apoptosis and release of inflammatory factors were alleviated by pretreatment of the Jun N-terminal kinase (JNK) inhibitor SP600125 or the p38 MAPK inhibitor SB203580, but not by the extracellular signal-regulated kinase (ERK) inhibitor PD98059 in UCP2-overexpressing mice. On the other hand, LPS-induced alveolar epithelial cell death and inflammation were attenuated by genipin. In conclusion, UCP2 increased susceptibility to LPS-induced cell death and pulmonary inflammation, most likely via ATP depletion and activation of MAPK signaling following ALI in mice. PMID:27057102

  12. Fibrinogen γ' increases the sensitivity to activated protein C in normal and factor V Leiden plasma.

    PubMed

    Omarova, Farida; Uitte de Willige, Shirley; Simioni, Paolo; Ariëns, Robert A S; Bertina, Rogier M; Rosing, Jan; Castoldi, Elisabetta

    2014-08-28

    Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance. © 2014 by The American Society of Hematology.

  13. Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

    PubMed Central

    Yamashiro, Shigeko; Yamakita, Yoshihiko; Ono, Shoichiro; Matsumura, Fumio

    1998-01-01

    Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery. PMID:9571235

  14. KRIT1 protein depletion modifies endothelial cell behavior via increased vascular endothelial growth factor (VEGF) signaling.

    PubMed

    DiStefano, Peter V; Kuebel, Julia M; Sarelius, Ingrid H; Glading, Angela J

    2014-11-21

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1(+/-) mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.

  15. A soluble bone morphogenetic protein type IA receptor increases bone mass and bone strength.

    PubMed

    Baud'huin, Marc; Solban, Nicolas; Cornwall-Brady, Milton; Sako, Dianne; Kawamoto, Yoshimi; Liharska, Katia; Lath, Darren; Bouxsein, Mary L; Underwood, Kathryn W; Ucran, Jeffrey; Kumar, Ravindra; Pobre, Eileen; Grinberg, Asya; Seehra, Jasbir; Canalis, Ernesto; Pearsall, R Scott; Croucher, Peter I

    2012-07-24

    Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.

  16. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs.

    PubMed

    Fain, John N; Company, Joseph M; Booth, Frank W; Laughlin, M Harold; Padilla, Jaume; Jenkins, Nathan T; Bahouth, Suleiman W; Sacks, Harold S

    2013-10-01

    Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16-20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Increased expression of fatty acid binding protein 4 in preeclamptic Placenta and its relevance to preeclampsia.

    PubMed

    Yan, Yuying; Peng, Huilian; Wang, Peng; Wang, Hanzhi; Dong, Minyue

    2016-03-01

    The aim of this investigation was to determine the expression of fatty acid binding protein 4 (FABP4) in the placenta from women with preeclampsia and normal pregnancy, and to delineate the regulatory effects on thophoblast cell by FABP4. We determined the expression of FABP4 by real-time polymerase chain reaction (PCR) for messenger ribonucleic acid (mRNA) or enzyme-linked immunesorbent assay (ELISA) and Western blotting for protein. Small interference of ribonucleic acid (siRNA) and specific FABP4 inhibitor were used to inhibit FABP4. The proliferation, migration and invasion of trophoblastic cells (Swan-71 and Jar) were evaluated with cell counting kit-8, wound-healing test and transwell analysis respectively. We found the expression of FABP4 was significantly higher in the placenta of preeclamptic women than that of women with normal pregnancy (t = 4.244, P < 0.001 for mRNA; t = 4.536, P < 0.001 for protein). FABP4 siRNA significantly reduced the proliferation of trophoblasts (P < 0.001). The specific inhibition of FABP4 inhibited the proliferation of trophoblasts in a dose-dependent manner (P < 0.001) and the inhibitory effect increased as the concentration of inhibitor increased. FABP4 siRNA and specific inhibitor significantly decreased the migration (P < 0.001) and invasion (P < 0.001) of trophoblasts. We concluded the increase in placental FABP4 expression in preeclampsia may affect the function of trophoblast, and this increase may have a role in the pathogenesis of preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Ischaemic preconditioning preferentially increases protein S-nitrosylation in subsarcolemmal mitochondria

    PubMed Central

    Sun, Junhui; Nguyen, Tiffany; Aponte, Angel M.; Menazza, Sara; Kohr, Mark J.; Roth, David M.; Patel, Hemal H.; Murphy, Elizabeth; Steenbergen, Charles

    2015-01-01

    Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischaemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signalling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analysed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared with IFM (1.33 ± 0.19, ratio of SNO content Perf-SSM vs. Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.72 ± 0.17 and 1.07 ± 0.04, ratio of SNO content IPC-SSM vs. Perf-IFM, and IPC-IFM vs. Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3, and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3−/− mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM may be the preferential target of sarcolemmal signalling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in IPC-induced cardioprotection. PMID:25694588

  19. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

    PubMed Central

    Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

    2013-01-01

    Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

  20. Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

    PubMed Central

    Pan, Keyao; Long, Jinxue; Sun, Haoxin; Tobin, Gregory J.; Nara, Peter L.

    2010-01-01

    The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R2 > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9405-4) contains supplementary material, which is available to authorized users. PMID:21086120

  1. Extending CATH: increasing coverage of the protein structure universe and linking structure with function.

    PubMed

    Cuff, Alison L; Sillitoe, Ian; Lewis, Tony; Clegg, Andrew B; Rentzsch, Robert; Furnham, Nicholas; Pellegrini-Calace, Marialuisa; Jones, David; Thornton, Janet; Orengo, Christine A

    2011-01-01

    CATH version 3.3 (class, architecture, topology, homology) contains 128,688 domains, 2386 homologous superfamilies and 1233 fold groups, and reflects a major focus on classifying structural genomics (SG) structures and transmembrane proteins, both of which are likely to add structural novelty to the database and therefore increase the coverage of protein fold space within CATH. For CATH version 3.4 we have significantly improved the presentation of sequence information and associated functional information for CATH superfamilies. The CATH superfamily pages now reflect both the functional and structural diversity within the superfamily and include structural alignments of close and distant relatives within the superfamily, annotated with functional information and details of conserved residues. A significantly more efficient search function for CATH has been established by implementing the search server Solr (http://lucene.apache.org/solr/). The CATH v3.4 webpages have been built using the Catalyst web framework.

  2. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis.

    PubMed

    Steelman, Zachary; Meng, Zhaokai; Traverso, Andrew J; Yakovlev, Vladislav V

    2015-05-01

    Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis. Typical raw output of a 2-stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross).

  3. Deletion of PTEN produces deficits in conditioned fear and increases fragile X mental retardation protein.

    PubMed

    Lugo, Joaquin N; Smith, Gregory D; Morrison, Jessica B; White, Jessika

    2013-11-15

    The phosphatase and tensin homolog detected on chromosome 10 (PTEN) gene product modulates activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The PI3K pathway has been found to be involved in the regulation of the fragile X mental retardation protein, which is important for long-term depression and in the formation of new memories. We used delayed fear conditioning and trace fear conditioning to determine learning and memory deficits in neuron subset-specific Pten (NS-Pten) conditional knockout (KO) mice. We found that NS-Pten KO mice had deficits in contextual learning and trace conditioning, but did not have deficits in the ability to learn a conditioned stimulus. Furthermore, we found increased levels in the total and phosphorylated forms of the fragile X mental retardation protein (FMRP) in the hippocampus of NS-Pten KO mice.

  4. T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

    PubMed Central

    Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A.; Edwards, David M.; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K.; Pickup, David J.; Lewinsohn, David M.; Gold, Marielle C.; Wong, Scott W.; Sacha, Jonah B.; Slifka, Mark K.; Früh, Klaus

    2014-01-01

    Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

  5. Increased response to morphine in mice lacking protein kinase C epsilon

    PubMed Central

    Newton, P. M.; Kim, J. A.; McGeehan, A. J.; Paredes, J. P.; Chu, K.; Wallace, M. J.; Roberts, A. J.; Hodge, C. W.; Messing, R. O.

    2014-01-01

    The protein kinase C (PKC) family of serine–threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCε have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCε null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCε null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of μ-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCε as a key regulator of opiate sensitivity in mice. PMID:16899053

  6. Increased expression of Dock180 protein in the noninfarcted myocardium in rats.

    PubMed

    Liu, Xiao-Lan; Li, Gang; Wang, Zhi-Hua; Zhao, Wen-Ju; Wang, Li-Ping

    2013-03-01

    The integrin β1 subunit and its downstream molecule focal adhesion kinase have been identified as critical molecules for the inhibition of postinfarction cardiac remodeling, ischemic cardiomyopathy, and heart failure. However, as a component of the integrin pathway, it is still unclear whether Dock180 (dedicator of cytokinesis 1) protein is expressed in the noninfarcted myocardium of the peri-infarct zones. In this study, experimental myocardial infarction (MI) and sham-operation (sham) models were established in Sprague Dawley rats and the expression of Dock180 protein in the myocardium of the sham group and in the noninfarcted myocardium of the peri-infarct zones of the MI group was detected by Western blot technique. The Dock180 protein expression in the myocardium was as follows: postsham 24-hour group, 0.10 ± 0.04 (n = 8); post-MI 24-hour group, 0.13 ± 0.03 (n = 8); postsham 12-week group, 0.11 ± 0.05 (n = 8); and post-MI 12-week group 0.17 ± 0.04 (n = 8). The Dock180 protein expression in the myocardium in the post-MI 12-week group was significantly higher than that in the postsham 12-week group (p = 0.019), in the postsham 24-hour group (p = 0.004), and in the post-MI 24-hour group (p = 0.040). We conclude that Dock180 protein is expressed in the myocardium in rats. Furthermore, its expression is significantly increased in the noninfarcted myocardium of the peri-infarct zones.

  7. DHEA increases epithelial markers and decreases mesenchymal proteins in breast cancer cells and reduces xenograft growth.

    PubMed

    Colín-Val, Zaira; González-Puertos, Viridiana Yazmín; Mendoza-Milla, Criselda; Gómez, Erika Olivia; Huesca-Gómez, Claudia; López-Marure, Rebeca

    2017-10-15

    Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated. In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu(-)/nu(-)) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated. We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation. In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Template-directed protein folding into a metastable state of increased activity.

    PubMed

    Flecker, P

    1995-09-01

    The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P'1Ser-->Pro variant in the trypsin-reactive subdomain differ before and after refolding on trypsin-Sepharose. A heterogenous and thermodynamically dominant population of conformers was attained in solution. However, the enzyme-inhibitory activity of the variant was dramatically increased in the presence of trypsin-Sepharose and a stoichiometric ratio of the two subdomains was obtained as expected for a single conformation. The subsequent return for the initial mixture of conformers in solution reveals a high kinetic barrier late in the folding process. The template facilitates folding kinetically, as shown by a rate acceleration of more than four orders of magnitude. The final state was also the thermodynamically favoured one on the template, due to its increased affinity for the enzyme. The long-range effects on folding kinetics and the partial activity, and the absence of free sulfhydryl groups after refolding in solution indicate rearrangements between closely related conformers late in folding. The importance of minor structural distortions in immobilized trypsin suggests a close structural analogy between the final and the transition state of protein folding.

  9. Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

    PubMed

    Quinet, E M; Agellon, L B; Kroon, P A; Marcel, Y L; Lee, Y C; Whitlock, M E; Tall, A R

    1990-02-01

    Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

  10. Oral supplementation with whey proteins increases plasma glutathione levels of HIV-infected patients.

    PubMed

    Micke, P; Beeh, K M; Schlaak, J F; Buhl, R

    2001-02-01

    HIV infection is characterized by an enhanced oxidant burden and a systemic deficiency of the tripeptide glutathione (GSH), a major antioxidant. The semi-essential amino acid cysteine is the main source of the free sulfhydryl group of GSH and limits its synthesis. Therefore, different strategies to supplement cysteine supply have been suggested to increase glutathione levels in HIV-infected individuals. The aim of this study was to evaluate the effect of oral supplementation with two different cysteine-rich whey protein formulas on plasma GSH levels and parameters of oxidative stress and immune status in HIV-infected patients. In a prospective double blind clinical trial, 30 patients (25 male, 5 female; mean age (+/- SD) 42 +/- 9.8 years) with stable HIV infection (221 +/- 102 CD4 + lymphocytes L-1) were randomized to a supplemental diet with a daily dose of 45 g whey proteins of either Protectamin (Fresenius Kabi, Bad Hamburg, Germany) or Immunocal (Immunotec, Vandreuil, Canada) for two weeks. Plasma concentrations of total, reduced and oxidized GSH, superoxide anion (O2-) release by blood mononuclear cells, plasma levels of TNF-alpha and interleukins 2 and 12 were quantified with standard methods at baseline and after therapy. Pre-therapy, plasma GSH levels (Protectamin: 1.92 +/- 0.6 microM; Immunocal: 1.98 +/- 0.9 microM) were less than normal (2.64 +/- 0.7 microM, P = 0.03). Following two weeks of oral supplementation with whey proteins, plasma GSH levels increased in the Protectamin group by 44 +/- 56% (2.79 +/- 1.2 microM, P = 0.004) while the difference in the Immunocal group did not reach significance (+ 24.5 +/- 59%, 2.51 +/- 1.48 microM, P = 0.43). Spontaneous O2- release by blood mononuclear cells was stable (20.1 +/- 14.2 vs. 22.6 +/- 16.1 nmol h-1 10-6 cells, P = 0.52) whereas PMA-induced O2- release decreased in the Protectamin group (53.7 +/- 19 vs. 39.8 +/- 18 nmol h-1 10-6 cells, P = 0.04). Plasma concentrations of TNF-alpha and interleukins 2 and

  11. Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate.

    PubMed

    Eckert, G E; Greene, L W; Carstens, G E; Ramsey, W S

    1999-01-01

    The Cu status of mature, crossbred ewes fed two sources (CuSO4 vs. Cu proteinate) and three levels (10, 20, or 30 mg/kg) of dietary Cu was determined in a 73-d feeding trial. Ewes (n = 30) were fed a basal diet containing rice meal feed, cottonseed hulls, cottonseed meal, meat and bone meal, cracked corn, and vitamin-mineral supplements at 2.5% of BW to meet NRC requirements for protein, energy, macrominerals, and microminerals, excluding Cu. The basal diet contained 5 mg/kg Cu, 113 mg/kg Fe, .1 mg/kg Mo, and .17% S. Copper sulfate or Cu proteinate was added to the basal diet to supply 10, 20, or 30 mg/kg of dietary copper in a 2x3 factorial arrangement of treatments. Ewes were housed in 3.7- x 9.1-m pens in an open-sided barn. Blood samples were collected on d 28 and 73. Ewes were slaughtered on d 74, and liver and other tissues were collected to determine Cu concentrations. An interaction (P = .08) occurred between source and level for liver Cu. The interaction existed due to an increase in liver Cu concentrations when ewes were fed increasing dietary Cu from CuSO4 but not when fed Cu proteinate diets. There was no source x level interaction (P>.10) for the blood constituents measured. On d 73, plasma ceruloplasmin activity was greater (P<.05) in ewes fed Cu proteinate than in those fed CuSO4 (33.1 vs. 26.8 microM x min(-1) x L(-1)). Increasing the concentration of dietary Cu did not affect (P>.10) plasma ceruloplasmin. Packed cell volume (PCV), red blood cell count (RBC), white blood cell count, whole blood hemoglobin (wHb), plasma hemoglobin, and plasma Cu were similar between sources of Cu. Ewes fed 20 mg/kg Cu had lower (P<.05) PCV, RBC, and wHb than those fed 10 or 30 mg/kg Cu diets. Feeding up to 30 mg/kg Cu from these sources did not cause an observable Cu toxicity during the 73-d period.

  12. Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

    PubMed Central

    Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

    2016-01-01

    Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

  13. Iron Regulatory Protein-2 Knockout Increases Perihematomal Ferritin Expression and Cell Viability after Intracerebral Hemorrhage

    PubMed Central

    Chen, Mai; Awe, Olatilewa O.; Chen-Roetling, Jing; Regan, Raymond F.

    2010-01-01

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls three days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at three days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries. PMID:20399759

  14. Halobacterial nano vesicles displaying murine bactericidal permeability-increasing protein rescue mice from lethal endotoxic shock

    PubMed Central

    Balakrishnan, Arjun; DasSarma, Priya; Bhattacharjee, Oindrilla; Kim, Jong Myoung; DasSarma, Shiladitya; Chakravortty, Dipshikha

    2016-01-01

    Bactericidal/permeability-increasing protein (BPI) had been shown to possess anti-inflammatory and endotoxin neutralizing activity by interacting with LPS of Gram-negative bacteria. The current study examines the feasibility of using murine BPI (mBPI) expressed on halophilic Archaeal gas vesicle nanoparticles (GVNPs) for the treatment of endotoxemia in high-risk patients, using a murine model of D-galactosamine-induced endotoxic shock. Halobacterium sp. NRC-1was used to express the N-terminal 199 amino acid residues of mBPI fused to the GVNP GvpC protein, and bound to the surface of the haloarchaeal GVNPs. Our results indicate that delivery of mBPIN-GVNPs increase the survival rate of mice challenged with lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine. Additionally, the mBPIN-GVNP-treated mice displayed reduced symptoms of inflammation, including inflammatory anemia, recruitment of neutrophils, liver apoptosis as well as increased pro-inflammatory serum cytokine levels. PMID:27646594

  15. Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage.

    PubMed

    Chen, Mai; Awe, Olatilewa O; Chen-Roetling, Jing; Regan, Raymond F

    2010-06-14

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls 3 days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at 3 days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries.

  16. Dry Eye Symptoms Are Increased in Mice Deficient in Phospholipid Transfer Protein (PLTP)

    PubMed Central

    Setälä, Niko L.; Metso, Jari; Jauhiainen, Matti; Sajantila, Antti; Holopainen, Juha M.

    2011-01-01

    In the tear fluid the outermost part facing the tear–air interface is composed of lipids preventing evaporation of the tears. Phospholipid transfer protein (PLTP) mediates phospholipid transfer processes between serum lipoproteins and is also a normal component of human tears. To study whether PLTP plays any functional role in tear fluid we investigated PLTP-deficient mice, applying functional and morphologic analyses under normal housing and experimentally induced dry eye conditions. Aqueous tear fluid production, corneal epithelial morphology, barrier function, and occludin expression were assessed. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression were shown. These pathologic signs were worsened by experimentally induced dry eye both in wild-type and PLTP knock-out mice. Deficiency in the production of tear PLTP in mice is accompanied by corneal epithelial damage, a feature that is typical in human dry eye syndrome (DES). To complement animal experiments we collected tear fluid from human dry eye patients as well as healthy control subjects. Increased tear fluid PLTP activity was observed among DES patients. In conclusion, the presence of PLTP in tear fluid appears to be essential for maintaining a healthy and functional ocular surface. Increased PLTP activity in human tear fluid in DES patients suggests an ocular surface protective role for this lipid transfer protein. PMID:21514421

  17. The evaluation of increase in hemodialysis frequency on C-reactive protein levels and nutritional status.

    PubMed

    Rashidi, Ali Akbar; Soleimani, Ali Reza; Nikoueinejad, Hassan; Sarbolouki, Shokooh

    2013-03-16

    Malnutrition and inflammation are the most important causes of cardiovascular disease in hemodialysis patients. This study was conducted to evaluate the effect of increase in hemodialysis frequency on C-reactive protein (CRP) level and nutritional markers in contrast to previous routine method. 18 hemodialysis patients with a mean age of 53±16 years were randomly selected in this before-and-after clinical trial. The patients under a standard hemodialysis of 3 times/4 h per week were converted to 4 times/4 h for a period of 6 weeks. The CRP, albumin, triglyceride, total cholesterol, LDL, HDL serum levels, anthropometric indices and 24-h diet recall intake was assessed before and after of the period. The data were analyzed using paired t-test, and P-value less than 0.05 was considered significant. All patients completed the study. Mean weight, body mass index and serum albumin increased while serum CRP level decreased significantly after the intervention (P<0.03). Triglyceride, total cholesterol, LDL, HDL, as well as energy, protein and fat intake had no significant change before and after the study. Increase in dialysis frequency decreased systemic inflammation and improved the nutritional state of hemodialysis patients. Therefore, it may decrease the risk of cardiovascular events in these patients.

  18. Peptidoglycan recognition protein-peptidoglycan complexes increase monocyte/macrophage activation and enhance the inflammatory response.

    PubMed

    De Marzi, Mauricio C; Todone, Marcos; Ganem, María B; Wang, Qian; Mariuzza, Roy A; Fernández, Marisa M; Malchiodi, Emilio L

    2015-07-01

    Peptidoglycan recognition proteins (PGRP) are pattern recognition receptors that can bind or hydrolyse peptidoglycan (PGN). Four human PGRP have been described: PGRP-S, PGRP-L, PGRP-Iα and PGRP-Iβ. Mammalian PGRP-S has been implicated in intracellular destruction of bacteria by polymorphonuclear cells, PGRP-Iα and PGRP-Iβ have been found in keratinocytes and epithelial cells, and PGRP-L is a serum protein that hydrolyses PGN. We have expressed recombinant human PGRP and observed that PGRP-S and PGRP-Iα exist as monomer and disulphide dimer proteins. The PGRP dimers maintain their biological functions. We detected the PGRP-S dimer in human serum and polymorphonuclear cells, from where it is secreted after degranulation; these cells being a possible source of serum PGRP-S. Recombinant PGRP do not act as bactericidal or bacteriostatic agents in the assayed conditions; however, PGRP-S and PGRP-Iα cause slight damage in the bacterial membrane. Monocytes/macrophages increase Staphylococcus aureus phagocytosis in the presence of PGRP-S, PGRP-Iα and PGRP-Iβ. All PGRP bind to monocyte/macrophage membranes and are endocytosed by them. In addition, all PGRP protect cells from PGN-induced apoptosis. PGRP increase THP-1 cell proliferation and enhance activation by PGN. PGRP-S-PGN complexes increase the membrane expression of CD14, CD80 and CD86, and enhance secretion of interleukin-8, interleukin-12 and tumour necrosis factor-α, but reduce interleukin-10, clearly inducing an inflammatory profile.

  19. Association analysis of bovine bactericidal/permeability-increasing protein gene polymorphisms with somatic cell score in Holstein cattle

    USDA-ARS?s Scientific Manuscript database

    Bactericidal/permeability-increasing (BPI) protein is expressed primarily in bovine neutrophils and epithelial cells and functions as a binding protein of bacterial lipopolysaccharide produced by Gram-negative bacteria. The protein is important in host defense against bacterial infections and may pl...

  20. Systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies against bactericidal/permeability increasing protein.

    PubMed

    Fukuhara, Atsuro; Tanino, Yoshinori; Sato, Suguru; Ishii, Taeko; Nikaido, Takefumi; Kanazawa, Kenya; Saito, Junpei; Ishida, Takashi; Kanno, Makoto; Watanabe, Tsuyoshi; Munakata, Mitsuru

    2013-01-01

    Myeloperoxidase- and proteinase 3-anti-neutrophil cytoplasmic antibodies (ANCAs) are often negative in cases in which systemic vasculitis is highly suspected. We herein present a case of bactericidal/permeability increasing protein (BPI)-ANCA-positive systemic vasculitis. This case highlights the possible role of BPI-ANCA in the pathogenesis of systemic vasculitis as well as the possible use of BPI as a diagnostic tool. The accumulation of further case-based reports is expected to shed some light on the pathogesis of systemic vasculitis.

  1. Chronic SIV and Morphine treatment increases heat shock protein 5 expression at the synapse

    PubMed Central

    Pendyala, Gurudutt; Periyasamy, Palsamy; Callen, Shannon; Fox, Howard S.; Lisco, Steven J.; Buch, Shilpa J.

    2015-01-01

    The abuse of opiates such as morphine in synergy with HIV infection accelerates neurocognitive impairments and neuropathology in the CNS of HIV infected subjects, collectively referred to as HAND. To identify potential pathogenic markers associated with HIV and morphine in perturbing the synaptic architecture, we performed quantitative mass spectrometry proteomics on purified synaptosomes isolated from the caudate of two groups of rhesus macaques chronically infected with SIV differing by one regimen- morphine treatment. The up regulation of heat shock 70 kDa protein 5 in the SIV+morphine group points to increased cellular stress during SIV/Morphine interaction thus leading to CNS dysfunction. PMID:26037114

  2. Chronic SIV and morphine treatment increases heat shock protein 5 expression at the synapse.

    PubMed

    Pendyala, Gurudutt; Periyasamy, Palsamy; Callen, Shannon; Fox, Howard S; Lisco, Steven J; Buch, Shilpa J

    2015-10-01

    The abuse of opiates such as morphine in synergy with HIV infection accelerates neurocognitive impairments and neuropathology in the CNS of HIV-infected subjects, collectively referred to as HAND. To identify potential pathogenic markers associated with HIV and morphine in perturbing the synaptic architecture, we performed quantitative mass spectrometry proteomics on purified synaptosomes isolated from the caudate of two groups of rhesus macaques chronically infected with SIV differing by one regimen-morphine treatment. The upregulation of heat shock 70-kDa protein 5 in the SIV + morphine group points to increased cellular stress during SIV/morphine interaction thus leading to CNS dysfunction.

  3. NFATc1 phosphorylation by DYRK1A increases its protein stability

    PubMed Central

    Chen, Shuai; Sun, Qian; Zhang, Yuankai; Chen, Long; Sun, Xiulian

    2017-01-01

    NFATs are transcription factors involved in immune activation and tumor progression. Previous reports showed that DYRK1A suppressed NFATc2 transcriptional activity through phosphorylation. Nonetheless, our results showed that DYRK1A increased NFATc1/αA protein level and subsequent transcriptional activity. DYRK1A phosphorylation of NFATc1/αA at S261, S278, S403 and S409 interfered with NFATc1 ubiquitination and ubiquitin-proteasome degradation. Our results imply that DYRK1A is a positive kinase in regulation of NFATc1. PMID:28235034

  4. Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm

    PubMed Central

    Liu, Chen; Li, Shixue; Yue, Jing; Xiao, Wenhan; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

    2015-01-01

    Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality. PMID:26703573

  5. The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity.

    PubMed

    Fortier, N; Snyder, L M; Garver, F; Kiefer, C; McKenney, J; Mohandas, N

    1988-05-01

    In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta-subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

  6. Suilysin Stimulates the Release of Heparin Binding Protein from Neutrophils and Increases Vascular Permeability in Mice

    PubMed Central

    Chen, Shaolong; Xie, Wenlong; Wu, Kai; Li, Ping; Ren, Zhiqiang; Li, Lin; Yuan, Yuan; Zhang, Chunmao; Zheng, Yuling; Lv, Qingyu; Jiang, Hua; Jiang, Yongqiang

    2016-01-01

    Most of the deaths that occurred during two large outbreaks of Streptococcus suis infections in 1998 and 2005 in China were caused by streptococcal toxic shock syndrome (STSS), which is characterized by increased vascular permeability. Heparin-binding protein (HBP) is thought to mediate the vascular leakage. The purpose of this study was to investigate the detailed mechanism underlying the release of HBP and the vascular leakage induced by S. suis. Significantly higher serum levels of HBP were detected in Chinese patients with STSS than in patients with meningitis or healthy controls. Suilysin (SLY) is an exotoxin secreted by the highly virulent strain 05ZYH33, and it stimulated the release of HBP from the polymorphonuclear neutrophils and mediated vascular leakage in mice. The release of HBP induced by SLY was caused by a calcium influx-dependent degranulation. Analyses using a pharmacological approach revealed that the release of HBP induced by SLY was related to Toll-like receptor 4, p38 mitogen-activated protein kinase, and the 1-phosphatidylinositol 3-kinase pathway. It was also dependent on a G protein-coupled seven-membrane spanning receptor. The results of this study provide new insights into the vascular leakage in STSS associated with non-Group A streptococci, which could lead to the discovery of potential therapeutic targets for STSS associated with S. suis. PMID:27617009

  7. Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

    PubMed

    Meghraoui, Alaeddine; Schiavolin, Lionel; Allaoui, Abdelmounaaïm

    2014-07-01

    Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence.

  8. Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

    PubMed Central

    Karamanlidis, Georgios; Lee, Chi Fung; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Suthammarak, Wichit; Gong, Guohua; Sedensky, Margaret M.; Morgan, Philip G.; Wang, Wang; Tian, Rong

    2013-01-01

    Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target. PMID:23931755

  9. Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

    PubMed Central

    Maass, Dirk; Arango, Jacobo; Wüst, Florian; Beyer, Peter; Welsch, Ralf

    2009-01-01

    Background As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. Methology/Principal Findings In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. Conclusions The sequestration of carotenoids into crystals can be driven by the functional overexpression of one

  10. Dietary psyllium fiber increases intestinal heat shock protein 25 expression in mice.

    PubMed

    Ogata, Miyuki; Van Hung, Tran; Tari, Hiroyuki; Arakawa, Teruaki; Suzuki, Takuya

    2017-03-01

    Heat shock proteins (HSPs) protect intestinal epithelial cell function, integrity and viability against many forms of stress. We hypothesized that dietary fibers (DFs) in the diet may increase HSP expression, since DFs are known to exhibit beneficial effects on intestinal health. The present study investigated the regulation of intestinal HSP expression by DFs, particularly psyllium fiber. Feeding psyllium fiber for 5 d increased HSP25 expression, but not HSP32 and HSP70 expression in the jejunum, ileum, and colon of mice at both the protein and mRNA levels. The increases in HSP25 expression did not correlate with cecal organic acid production by microbial fermentation. The water-insoluble fraction of psyllium fiber largely contributed to the induction of HSP25 expression, but feeding with other water-insoluble DFs from beet, wheat, and oats failed to induce intestinal HSP25 expression. Although the water-holding capacity of psyllium fiber was much higher than those of the other water-insoluble DFs examined, the increase in HSP25 expression induced by feeding polycarbophil, which possesses a high water-holding capacity similar to that of psyllium fiber, was much lower than that induced by psyllium fiber. Finally, induction of malondialdehyde production by hydrogen peroxide, an oxidant, in the colon of mice fed psyllium fiber was lower than that in mice fed with the control diets. Taken together, feeding psyllium fiber, especially the water-insoluble fraction, increases intestinal HSP25 expression and suppresses oxidant-induced malondialdehyde production. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Fermentation of rapeseed meal, sunflower meal and faba beans in combination with wheat bran increases solubility of protein and phosphorus.

    PubMed

    Poulsen, Hanne Damgaard; Blaabjerg, Karoline

    2017-01-01

    To increase self-supply of protein and phosphorus (P) in European pig and poultry diets and reduce nitrogen (N) and P excretion, attention is directed to approaches increasing protein and P digestibility of rapeseed, sunflower and faba beans. Wheat bran is rich in enzymes degrading and solubilizing protein and phytate. Herein, solubilization of protein, N and P was investigated when increasing ratios of wheat bran were fermented with rapeseed meal (RSM), sunflower meal (SFM), faba beans (FB) or a combination of these (RSM/SFM/FB). Protein, N and P solubility was greater, for all mixtures, the more wheat bran was included and the longer the mixtures were fermented. The increase in N (FB > RSM/SFM/FB > SFM > RSM) and protein solubility (RSM/SFM/FB > RSM > SFM > FB) was greatest from day 0 to day 3 and thereafter limited, whereas P solubility increased during the whole period (5 days; FB > RSM/SFM/FB > SFM > RSM). In general, FB showed the highest solubility and highest increase in N and P solubility, while RSM showed the highest protein solubility and RSM/SFM/FB the highest increase in protein solubility. Fermentation of RSM, SFM, FB and RSM/SFM/FB without or with wheat bran uncovers a potential for increased protein and P digestibility and thereby reduced N and P excretion from pigs and poultry. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  12. O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

    PubMed Central

    Guinez, Céline; Filhoulaud, Gaëlle; Rayah-Benhamed, Fadila; Marmier, Solenne; Dubuquoy, Céline; Dentin, Renaud; Moldes, Marthe; Burnol, Anne-Françoise; Yang, Xiaoyong; Lefebvre, Tony; Girard, Jean; Postic, Catherine

    2011-01-01

    OBJECTIVE Carbohydrate-responsive element–binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. RESEARCH DESIGN AND METHODS O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBPOG in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. RESULTS Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBPOG in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice

  13. Circadian Misalignment Increases C-Reactive Protein and Blood Pressure in Chronic Shift Workers.

    PubMed

    Morris, Christopher J; Purvis, Taylor E; Mistretta, Joseph; Hu, Kun; Scheer, Frank A J L

    2017-03-01

    Shift work is a risk factor for inflammation, hypertension, and cardiovascular disease. This increased risk cannot be fully explained by classical risk factors. Shift workers' behavioral and environmental cycles are typically misaligned relative to their endogenous circadian system. However, there is little information on the impact of acute circadian misalignment on cardiovascular disease risk in shift workers, independent of differences in work stress, food quality, and other factors that are likely to differ between night and day shifts. Thus, our objectives were to determine the independent effect of circadian misalignment on 24-h high-sensitivity C-reactive protein (hs-CRP; a marker of systemic inflammation) and blood pressure levels-cardiovascular disease risk factors-in chronic shift workers. Chronic shift workers undertook two 3-day laboratory protocols that simulated night work, comprising 12-hour inverted behavioral and environmental cycles (circadian misalignment) or simulated day work (circadian alignment), using a randomized, crossover design. Circadian misalignment increased 24-h hs-CRP by 11% ( p < 0.0001). Circadian misalignment increased 24-h systolic blood pressure (SBP) and diastolic blood pressure (DBP) by 1.4 mmHg and 0.8 mmHg, respectively (both p ≤ 0.038). The misalignment-mediated increase in 24-h SBP was primarily explained by an increase in SBP during the wake period (+1.7 mmHg; p = 0.017), whereas the misalignment-mediated increase in 24-h DBP was primarily explained by an increase in DBP during the sleep opportunity (+1.8 mmHg; p = 0.005). Circadian misalignment per se increases hs-CRP and blood pressure in shift workers. This may help explain the increased inflammation, hypertension, and cardiovascular disease risk in shift workers.

  14. Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

    PubMed Central

    de la Cruz, J; Vioque, A

    2001-01-01

    tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628

  15. Learning selectively increases protein kinase C substrate phosphorylation in specific regions of the chick brain.

    PubMed Central

    Sheu, F S; McCabe, B J; Horn, G; Routtenberg, A

    1993-01-01

    The effect of imprinting, an early form of exposure learning, on the phosphorylation state of the protein kinase C substrates myristoylated alanine-rich C-kinase substrate (MARCKS) and protein F1/43-kDa growth-associated protein (F1/GAP-43) was studied in two regions of the chick forebrain. One region, the intermediate and medial part of the hyperstriatum ventrale (IMHV), is probably a site of long-term memory; the other, the wulst, contains somatic sensory and visual projection areas. After imprinting, a significant increase in MARCKS protein phosphorylation was observed in the left IMHV but not the right IMHV. No significant alteration in F1/GAP-43 was observed in IMHV. MARCKS was resolved into two acidic components of pI approximately 5.0 and approximately 4.0. Phosphorylation of the pI approximately 5.0 MARCKS but not the pI approximately 4.0 MARCKS was significantly altered by imprinting. The partial correlation between preference score (an index of learning) and phosphorylation, holding constant the effect of approach activity during training, was significant only for the pI approximately 5.0 MARCKS in the left IMHV. A significant negative partial correlation between preference score and F1/GAP-43 phosphorylation in the right wulst was observed. Because the imprinting-induced alteration in MARCKS is selective with respect to phosphoprotein moiety, hemispheric location, and brain region, we propose that these alterations may be central to the learning process. Images Fig. 1 Fig. 2 Fig. 3 PMID:8464879

  16. Taste sensitivity for monosodium glutamate and an increased liking of dietary protein.

    PubMed

    Luscombe-Marsh, Natalie D; Smeets, Astrid J P G; Westerterp-Plantenga, Margriet S

    2008-04-01

    The aim of the present study was to determine individuals' taste threshold for monosodium glutamate (MSG) alone and in combination with inosine 5'-monophosphate (IMP-5) and to examine if this threshold was related to an increase in sensory properties (including pleasantness of taste) and/or to one's preference for dietary protein over carbohydrate and fat. Using the triangle tasting method, the taste threshold was determined for thirty-six women and twenty-four men. Thresholds varied from zero to infinite as determined using a clear soup with added MSG in the concentration range of 0.1 to 0.8 % (w/w) MSG. Subjects rated fourteen sensory properties of the soup and also their 'liking', 'eating frequency' and 'preference' of twenty-two common high-protein, high-carbohydrate and high-fat food items. The taste threshold (and therefore sensitivity) of MSG was lowered from 0.33 (sem 0.24) to 0.26 (sem 0.22) % MSG when 0.25 % (w/w) IMP-5 was added. None of the sensory properties assessed was associated with the taste threshold of MSG +/- 0.25 % IMP-5 in the overall study population. However, the taste descriptor 'meatiness' was associated with the threshold data for individuals who could taste concentrations of protein were found to be related to the threshold of MSG +/- 0.25 % IMP-5. From this study population we conclude that the taste threshold of MSG in combination with IMP-5 does appear to predict one's 'liking' of as well as 'preference' for high-protein foods.

  17. Modeling the contribution of individual proteins to mixed skeletal muscle protein synthetic rates over increasing periods of label incorporation.

    PubMed

    Miller, Benjamin F; Wolff, Christopher A; Peelor, Fredrick F; Shipman, Patrick D; Hamilton, Karyn L

    2015-03-15

    Advances in stable isotope approaches, primarily the use of deuterium oxide ((2)H2O), allow for long-term measurements of protein synthesis, as well as the contribution of individual proteins to tissue measured protein synthesis rates. Here, we determined the influence of individual protein synthetic rates, individual protein content, and time of isotopic labeling on the measured synthesis rate of skeletal muscle proteins. To this end, we developed a mathematical model, applied the model to an established data set collected in vivo, and, to experimentally test the impact of different isotopic labeling periods, used (2)H2O to measure protein synthesis in cultured myotubes over periods of 2, 4, and 7 days. We first demonstrated the influence of both relative protein content and individual protein synthesis rates on measured synthesis rates over time. When expanded to include 286 individual proteins, the model closely approximated protein synthetic rates measured in vivo. The model revealed a 29% difference in measured synthesis rates from the slowest period of measurement (20 min) to the longest period of measurement (6 wk). In support of these findings, culturing of C2C12 myotubes with isotopic labeling periods of 2, 4, or 7 days revealed up to a doubling of the measured synthesis rate in the shorter labeling period compared with the longer period of labeling. From our model, we conclude that a 4-wk period of labeling is ideal for considering all proteins in a mixed-tissue fraction, while minimizing the slowing effect of fully turned-over proteins. In addition, we advocate that careful consideration must be paid to the period of isotopic labeling when comparing mixed protein synthetic rates between studies.

  18. Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise

    PubMed Central

    Philp, Andrew; Schenk, Simon; Perez-Schindler, Joaquin; Hamilton, D Lee; Breen, Leigh; Laverone, Erin; Jeromson, Stewart; Phillips, Stuart M; Baar, Keith

    2015-01-01

    Abstract The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min−1; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg−1) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg−1) of l-[ring-13C6]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation. Key points Previous studies have shown that endurance exercise increases myofibrillar (MyoPS) and

  19. Electric field stimulation can increase protein synthesis in articular cartilage explants.

    PubMed

    MacGinitie, L A; Gluzband, Y A; Grodzinsky, A J

    1994-03-01

    It has been hypothesized that the electric fields associated with the dynamic loading of cartilage may affect its growth, remodeling, and biosynthesis. While the application of exogenous fields has been shown to modulate cartilage biosynthesis, it is not known what range of field magnitudes and frequencies can alter biosynthesis and how they relate to the magnitudes and frequencies of endogenous fields. Such information is necessary to understand and identify mechanisms by which fields may act on cartilage metabolism. In this study, incorporation of 35S-methionine was used as a marker for electric field-induced changes in chondrocyte protein synthesis in disks of cartilage from the femoropatellar groove of 1 to 2-week-old calves. The cartilage was stimulated sinusoidally at 1, 10, 100, 10(3), and 10(4) Hz with current densities of 10-30 mA/cm2. Incorporation was assessed in control disks maintained in the absence of applied current at 37, 41, and 43 degrees C. The possibility that applied currents would induce synthesis of the same stress proteins that are caused by heating or other mechanisms was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examination of gel fluorographs. Total radiolabel incorporation in cartilage that had been stimulated relative to incorporation in the controls increased with current density magnitudes greater than 10 mA/cm2. The increase was greatest at 100 Hz and 1 kHz, and it depended on the position on the joint surface from which the cartilage samples were taken. Together, these results suggest that endogenous electric fields could affect cartilage biosynthesis. Stress proteins were not induced at any current density when the electrodes were electrically connected but chemically isolated from the media by agarose bridges. Stress proteins were observed for disks incubated at temperatures greater than 39 degrees C (no field) and when the stimulating platinum electrodes were in direct contact with the media

  20. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  1. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

    PubMed

    Allonso, Diego; Andrade, Iamara S; Conde, Jonas N; Coelho, Diego R; Rocha, Daniele C P; da Silva, Manuela L; Ventura, Gustavo T; Silva, Emiliana M; Mohana-Borges, Ronaldo

    2015-12-01

    Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the

  2. Increased light intensity induces heat shock protein Hsp60 in coral species.

    PubMed

    Chow, Ari M; Ferrier-Pagès, Christine; Khalouei, Sam; Reynaud, Stéphanie; Brown, Ian R

    2009-09-01

    The effect of increased light intensity and heat stress on heat shock protein Hsp60 was examined in two coral species using a branched coral and a laminar coral, selected for their different resistance to environmental perturbation. Transient Hsp60 induction was observed in the laminar coral following either light or thermal stress. Sustained induction was observed when these stresses were combined. The branched coral exhibited comparatively weak transient Hsp60 induction after heat stress and no detectable induction following light stress, consistent with its susceptibility to bleaching in native environments compared to the laminar coral. Our observations also demonstrate that increased light intensity and heat stress exhibited a greater negative impact on the photosynthetic capacity of environmentally sensitive branched coral than the more resistant laminar coral. This supports a correlation between stress induction of Hsp60 and (a) ability to counter perturbation of photosynthetic capacity by light and heat stress and (b) resistance to environmentally induced coral bleaching.

  3. Increasing the Chemical‐Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent

    PubMed Central

    Göbl, Christoph; Resch, Moritz; Strickland, Madeleine; Hartlmüller, Christoph; Viertler, Martin

    2016-01-01

    Abstract The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical‐shift assignments, and data analysis that relies on well‐separated resonances. We present a covalent paramagnetic lanthanide‐binding tag (LBT) for increasing the chemical‐shift dispersion and facilitating the chemical‐shift assignment of challenging, repeat‐containing IDPs. Linkage of the DOTA‐based LBT to a cysteine residue induces pseudo‐contact shifts (PCS) for resonances more than 20 residues from the spin‐labeling site. This leads to increased chemical‐shift dispersion and decreased signal overlap, thereby greatly facilitating chemical‐shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat‐containing IDPs and low‐complexity regions. This results in improved efficiency for IDP analysis and binding studies. PMID:27763708

  4. Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body

    PubMed Central

    Gehring, Katrin B.; Heufelder, Karin; Depner, Harald; Kersting, Isabella; Sigrist, Stephan J.

    2017-01-01

    In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals. PMID:28437454

  5. Nitrous oxide plus isoflurane induces apoptosis and increases β-amyloid protein levels

    PubMed Central

    Zhen, Yu; Dong, Yuanlin; Wu, Xu; Xu, Zhipeng; Lu, Yan; Zhang, Yiying; Norton, David; Tian, Ming; Li, Shuren; Xie, Zhongcong

    2009-01-01

    Background Some anesthetics have been suggested to induce neurotoxicity including promotion of Alzheimer’s disease neuropathogenesis. Nitrous oxide and isoflurane are common anesthetics. Here, we set out to assess effects of nitrous oxide and/or isoflurane on apoptosis and β-amyloid (Aβ) levels in H4 human neuroglioma cells and primary neurons from naïve mice. Methods The cells or neurons were exposed to 70% nitrous oxide and/or 1% isoflurane for six hours. The cells or neurons and conditioned media were harvested at the end of the treatment. Caspase-3 activation, apoptosis, processing of amyloid precursor protein, and Aβ levels were determined. Results Treatment with a combination of 70% nitrous oxide and 1% isoflurane for six hours induced caspase-3 activation and apoptosis in H4 naïve cells and primary neurons from naïve mice. The 70% nitrous oxide plus 1% isoflurane, but neither alone, for six hours induced caspase-3 activation and apoptosis, and increased levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in H4-amyloid precursor protein cells. In addition, the nitrous oxide plus isoflurane-induced Aβ generation was reduced by a broad caspase inhibitor Z-VAD. Finally, the nitrous oxide plus isoflurane-induced caspase-3 activation was attenuated by γ-secretase inhibitor L-685,458, but potentiated by exogenously added Aβ. Conclusion These results suggest that common anesthetics nitrous oxide plus isoflurane may promote neurotoxicity by inducing apoptosis and increasing Aβ levels. The generated Aβ may further potentiate apoptosis to form another round of apoptosis and Aβ generation. More studies, especially the in vivo confirmation of these in vitro findings, are needed. PMID:19741497

  6. Low-Molecular-Weight Protein Tyrosine Phosphatase Predicts Prostate Cancer Outcome by Increasing the Metastatic Potential.

    PubMed

    Ruela-de-Sousa, Roberta R; Hoekstra, Elmer; Hoogland, A Marije; Queiroz, Karla C Souza; Peppelenbosch, Maikel P; Stubbs, Andrew P; Pelizzaro-Rocha, Karin; van Leenders, Geert J L H; Jenster, Guido; Aoyama, Hiroshi; Ferreira, Carmen V; Fuhler, Gwenny M

    2016-04-01

    Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. These findings implicate low

  7. Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition

    PubMed Central

    Meyers, Emily A.; Gobeske, Kevin T.; Bond, Allison M.; Jarrett, Jennifer C.; Peng, Chian-Yu; Kessler, John A.

    2015-01-01

    Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. PMID:26827654

  8. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

    PubMed

    Cruz-Teran, Carlos A; Carlin, Kevin B; Efimenko, Kirill; Genzer, Jan; Rao, Balaji M

    2016-08-30

    While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.

  9. A dramatic increase of C1q protein in the CNS during normal aging.

    PubMed

    Stephan, Alexander H; Madison, Daniel V; Mateos, José María; Fraser, Deborah A; Lovelett, Emilie A; Coutellier, Laurence; Kim, Leo; Tsai, Hui-Hsin; Huang, Eric J; Rowitch, David H; Berns, Dominic S; Tenner, Andrea J; Shamloo, Mehrdad; Barres, Ben A

    2013-08-14

    The decline of cognitive function has emerged as one of the greatest health threats of old age. Age-related cognitive decline is caused by an impacted neuronal circuitry, yet the molecular mechanisms responsible are unknown. C1q, the initiating protein of the classical complement cascade and powerful effector of the peripheral immune response, mediates synapse elimination in the developing CNS. Here we show that C1q protein levels dramatically increase in the normal aging mouse and human brain, by as much as 300-fold. This increase was predominantly localized in close proximity to synapses and occurred earliest and most dramatically in certain regions of the brain, including some but not all regions known to be selectively vulnerable in neurodegenerative diseases, i.e., the hippocampus, substantia nigra, and piriform cortex. C1q-deficient mice exhibited enhanced synaptic plasticity in the adult and reorganization of the circuitry in the aging hippocampal dentate gyrus. Moreover, aged C1q-deficient mice exhibited significantly less cognitive and memory decline in certain hippocampus-dependent behavior tests compared with their wild-type littermates. Unlike in the developing CNS, the complement cascade effector C3 was only present at very low levels in the adult and aging brain. In addition, the aging-dependent effect of C1q on the hippocampal circuitry was independent of C3 and unaccompanied by detectable synapse loss, providing evidence for a novel, complement- and synapse elimination-independent role for C1q in CNS aging.

  10. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    PubMed

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose.

  11. Protection against endotoxic shock by bactericidal/permeability-increasing protein in rats.

    PubMed Central

    Jin, H; Yang, R; Marsters, S; Ashkenazi, A; Bunting, S; Marra, M N; Scott, R W; Baker, J B

    1995-01-01

    Bactericidal/permeability-increasing protein (BPI) is a neutrophil primary granule protein that inhibits effects of LPS in vitro. The current study examined the effects of BPI on hemodynamics, mortality, and circulating endotoxin and cytokines in conscious rats with endotoxic shock. Catheters were implanted into the right femoral artery and vein. 1 d later, human recombinant BPI (10 mg/kg) or vehicle was intravenously injected immediately, 30 min, or 2 h after intravenous injection of LPS (7.5 mg/kg). Mean arterial pressure (MAP) and heart rate were monitored and blood was collected before and after injection. BPI given immediately or 30 min after LPS prevented the LPS-induced reduction in MAP at 4-8 h and markedly reduced mortality. BPI given 2 h after LPS injection had no protective effect. BPI treated immediately after LPS reduced the circulating levels of endotoxin and IL-6 but increased the circulating levels of TNF. We propose that BPI exerts its protective effect through a TNF-independent mechanism, by inhibiting endotoxin-stimulated production of IL-6. PMID:7706502

  12. Cholesterol-induced astrocyte activation is associated with increased amyloid precursor protein expression and processing.

    PubMed

    Avila-Muñoz, Evangelina; Arias, Clorinda

    2015-06-19

    Cholesterol is essential for maintaining lipid raft integrity and has been regarded as a crucial regulatory factor for amyloidogenesis in Alzheimer's disease (AD). The vast majority of studies on amyloid precursor protein (APP) metabolism and amyloid β-protein (Aβ) production have focused on neurons. The role of astrocytes remains largely unexplored, despite the presence of activated astrocytes in the brains of most patients with AD and in transgenic models of the disease. The role of cholesterol in Aβ production has been thoroughly studied in neurons and attributed to the participation of lipid rafts in APP metabolism. Thus, in this study, we analyzed the effect of cholesterol loading in astrocytes and analyzed the expression and processing of APP. We found that cholesterol exposure induced astrocyte activation, increased APP content, and enhanced the interaction of APP with BACE-1. These effects were associated with an enrichment of ganglioside GM1-cholesterol patches in the astrocyte membrane and with increased ROS production. GLIA 2015. © 2015 Wiley Periodicals, Inc.

  13. Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli.

    PubMed

    Matusheski, Nathan V; Juvik, John A; Jeffery, Elizabeth H

    2004-05-01

    Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.

  14. Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI) Expression in Intestinal Epithelium.

    PubMed

    Balakrishnan, Arjun; Chakravortty, Dipshikha

    2017-01-01

    As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP) that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI) is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn's Disease, Ulcerative colitis, and Infectious enteritis's. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri) whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC) did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

  15. Agouti-related protein increases food hoarding more than food intake in Siberian hamsters.

    PubMed

    Day, Diane E; Bartness, Timothy J

    2004-01-01

    Agouti-related protein (AgRP), an endogenous melanocortin 3/4 receptor antagonist, appears to play an important role in the control of food intake and energy balance because exogenous administration in rats and overexpression in mice result in hyperphagia and body mass gain. Furthermore, arcuate nucleus AgRP mRNA is increased with fasting in laboratory rats and mice and is decreased with refeeding. In Siberian hamsters, fasting also increases arcuate nucleus AgRP mRNA, but these animals increase food hoarding, rather than food intake with refeeding. Therefore, we tested whether exogenous AgRP increased food hoarding in this species. Hamsters were trained in a hoarding/foraging apparatus to run a programmed number of wheel revolutions to earn food pellets. Four doses of AgRP-(83-132) or vehicle were injected into the third ventricle at the beginning of the dark phase, and food hoarding, food intake, and foraging were measured at various time points subsequently. Overall, food hoarding was stimulated as much as 10 times more than food intake, and both responses occurred as early as 1 h after injection. Food hoarding was increased the greatest at the lowest dose (0.1 nmol), whereas food intake was increased the greatest at the second lowest dose (1 nmol). Food intake and especially food hoarding were increased up to seven days after the AgRP injections. Foraging was increased at all AgRP doses except the highest dose (100 nmol). These results suggest that AgRP triggers the search for food in this species, and once they find it, hoarding predominates over eating.

  16. Recycling of glucagon receptor to plasma membrane increases in adipocytes of obese rats by soy protein; implications for glucagon resistance.

    PubMed

    Velázquez-Villegas, Laura A; Tovar-Palacio, Claudia; Palacios-González, Berenice; Torres, Nimbe; Tovar, Armando R; Díaz-Villaseñor, Andrea

    2017-10-01

    Hyperglucagonemia contributes to hyperglycemia in type 2 diabetes (T2D). Previously, we have found that soy protein normalized fasting hyperglucagonemia in obese Zucker (fa/fa) rats, sensitizing the HSL-lipolytic signaling pathway in white adipose tissue (WAT), however the mechanism remains unknown. Zucker (fa/fa) rats were fed casein or soy protein diet in combination with soybean or coconut oil. Glucagon receptor (GR) was increased at the plasma membrane of adipocytes of rats fed soy protein compared to those fed casein, without changes in total GR abundance. The protein abundance of Rab4, a GTPase involved in GR fast recycling, was dramatically up-regulated in adipocytes of rats fed soy protein. The proportion of GR bound to Rab4 or to RAMP2, involved in promoting GR ligand-binding and G protein selectivity, increased when soy protein was combined with soybean oil as fat source. In rats fed soy protein with coconut oil, Rab11 levels, a protein involved in the slow recycling of GR, was also increased. Soy protein increases GR recycling to the membrane of adipocytes and its ligand-binding and G protein selectivity, suggesting, it could be used in T2D dietary treatment to reestablish glucagon sensitivity in WAT, leading to the regulation of circulating glucagon levels. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Protein kinase CK2 increases glutamatergic input in the hypothalamus and sympathetic vasomotor tone in hypertension.

    PubMed

    Ye, Zeng-You; Li, De-Pei; Li, Li; Pan, Hui-Lin

    2011-06-01

    Increased glutamatergic input in the paraventricular nucleus (PVN) is important for high sympathetic outflow in hypertension, but the associated molecular mechanisms remain unclear. Here, we determined the role of protein kinase CK2 (formerly casein kinase II) in increased N-methyl-d-aspartate receptor (NMDAR) activity in spinally projecting PVN neurons and sympathetic vasomotor tone in spontaneously hypertensive rats (SHRs). The selective CK2 inhibitors 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) significantly decreased the frequency of miniature EPSCs (mEPSCs) of labeled PVN neurons in SHRs but not in Wistar-Kyoto (WKY) normotensive rats. Also, DRB abolished the inhibitory effect of the NMDAR antagonist AP5 on the frequency of mEPSCs in SHRs. Treatment with DRB or TBB significantly reduced the amplitude of evoked NMDA-EPSCs but not AMPA-EPSCs in SHRs. Furthermore, DRB significantly decreased the firing activity of PVN neurons in SHRs but not in WKY rats. The membrane protein level of CK2α in the PVN, but not brainstem and prefrontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased CK2α level and the effects of DRB on mEPSCs and NMDA-EPSCs. In addition, intracerebroventricular injection of DRB not only significantly reduced blood pressure and lumbar sympathetic nerve discharges but also eliminated the inhibitory effect of AP5 microinjected into the PVN on sympathetic nerve activity in SHRs. Our findings suggest that augmented CK2 activity critically contributes to increased presynaptic and postsynaptic NMDAR activity in the PVN and elevated sympathetic vasomotor tone in essential hypertension.

  18. Resveratrol Administration Increases Transthyretin Protein Levels, Ameliorating AD Features: The Importance of Transthyretin Tetrameric Stability

    PubMed Central

    Santos, Luís Miguel; Rodrigues, Daniela; Alemi, Mobina; Silva, Sara Costa; Ribeiro, Carlos Alexandre; Cardoso, Isabel

    2016-01-01

    Previous in vivo work showed that resveratrol has beneficial effects in Alzheimer’s disease (AD) pathology, resulting in increased expression of transthyretin (TTR). TTR binds amyloid-beta (Aβ) peptide, avoiding its aggregation and toxicity, and is reduced in the cerebrospinal fluid (CSF) and plasma in AD. Further, resveratrol binds TTR, stabilizing the native TTR tetrameric structure. To further explore the mechanism of neuroprotection conferred by TTR in AD, resveratrol was administered in the diet to 5- to 8-month-old AD transgenic female mice carrying just 1 copy of the mouse TTR gene for 2 months. Effects in brain Aβ burden were evaluated by immunohistochemistry, and total brain Aβ levels by ELISA, showing a striking decrease in both parameters in treated animals. In addition, total brain lipoprotein-related receptor protein 1 (LRP1) levels were increased in treated animals, although its gene expression was unaltered. To further understand the mechanism(s) underlying such improvement in AD features, we measured TTR plasma levels, showing that TTR increased in resveratrol-treated mice, whereas liver TTR gene transcription was not altered. These results strengthen the stability hypothesis, which postulates that TTR is unstable in AD, leading to accelerated clearance and lower levels. Therefore, resveratrol, which stabilizes the TTR tetramer results in TTR-normalized clearance, increases the protein plasma levels. In turn, stabilized TTR binds more strongly to Aβ peptide, avoiding its aggregation. Our results represent a step forward in the understanding of the mechanism underlying TTR protection in AD and highlight the possibility of using TTR stabilization as a therapeutic target in AD. PMID:27385446

  19. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice.

    PubMed

    Kesby, James P; Hubbard, David T; Markou, Athina; Semenova, Svetlana

    2014-07-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. © 2012 The

  20. TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

    PubMed Central

    Geisberg, Joseph V.; Struhl, Kevin

    2000-01-01

    Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery. PMID:10669725

  1. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

    PubMed Central

    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  2. Resistance training increases heat shock protein levels in skeletal muscle of young and old rats.

    PubMed

    Murlasits, Zsolt; Cutlip, Robert G; Geronilla, Kenneth B; Rao, K Murali K; Wonderlin, William F; Alway, Stephen E

    2006-04-01

    Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (n=10) and old (30 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress

  3. Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise.

    PubMed

    Philp, Andrew; Schenk, Simon; Perez-Schindler, Joaquin; Hamilton, D Lee; Breen, Leigh; Laverone, Erin; Jeromson, Stewart; Phillips, Stuart M; Baar, Keith

    2015-09-15

    The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min(-1) ; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg(-1) ) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg(-1) ) of l-[ring-(13) C6 ]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  4. Increasing protein stability by polar surface residues : domain-wide consequences of interactions within a loop.

    SciTech Connect

    Pokkuluri, P. R.; Raffen, R.; Dieckman, L.; Boogaard, C.; Stevens, F. J.; Schiffer, M.; Biosciences Division; C. Boogaard

    2002-01-01

    We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.

  5. Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes

    PubMed Central

    Calderon-Dominguez, María; Sebastián, David; Fucho, Raquel; Weber, Minéia; Mir, Joan F.; García-Casarrubios, Ester; Obregón, María Jesús; Zorzano, Antonio; Valverde, Ángela M.; Serra, Dolors

    2016-01-01

    The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PMID:27438137

  6. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

    PubMed

    Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

    2017-02-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

  7. Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge.

    PubMed

    Bannerman, Douglas D; Paape, Max J; Hare, William R; Sohn, Eun Jung

    2003-10-01

    Several species of gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and various species of Enterobacter, are common mastitis pathogens. All of these bacteria are characterized by the presence of endotoxin or lipopolysaccharide (LPS) in their outer membrane. The bovine mammary gland is highly sensitive to LPS, and LPS has been implicated, in part, in the pathogenesis of gram-negative mastitis. Recognition of LPS is a key event in the innate immune response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). The objective of the current study was to determine whether LBP levels increased in the blood and mammary gland following LPS challenge. The left and right quarters of five midlactating Holstein cows were challenged with either saline or LPS (100 microg), respectively, and milk and blood samples collected. Basal levels of plasma and milk LBP were 38 and 6 microg/ml, respectively. Plasma LBP levels increased as early as 8 h post-LPS challenge and reached maximal levels of 138 microg/ ml by 24 h. Analysis of whey samples derived from LPS-treated quarters revealed an increase in milk LBP by 12 h. Similar to plasma, maximal levels of milk LBP (34 microg/ml) were detected 24 h following the initial LPS challenge. Increments in milk LBP levels paralleled a rise in soluble CD14 (sCD14) levels and initial rises in the levels of these proteins were temporally coincident with maximal neutrophil recruitment to the inflamed gland. Because LBP and sCD14 are known to enhance LPS-induced host cell activation and to facilitate detoxification of LPS, these data are consistent with a role for these molecules in mediating mammary gland responses to LPS.

  8. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  9. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  10. Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake

    PubMed Central

    Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

    2013-01-01

    Purpose The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Methods and Results Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of 57Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of 57FeSO4 or of 57Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. Conclusions The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis. PMID:23826373

  11. Soy germ protein concentrate diet decreased body fat weight and increased hindlimb muscle weight in rats.

    PubMed

    Kataoka, Hisashi; Saito, Sanshiro; Itoh, Atsushi; Matsuo, Tatsuhiro

    2012-01-01

    The purpose of this study was to investigate the effects of soy germ protein intake on body composition. Wistar rats were fed experimental diets for 16 weeks. These consisted of soy germ protein, soy protein, or casein. Abdominal adipose tissue weights significantly lower and hindlimb muscle weights were significantly higher in the soy germ protein group than in the casein group.

  12. Bactericidal/permeability-increasing protein preserves leukocyte functions after major liver resection.

    PubMed

    Wiezer, M J; Meijer, C; Sietses, C; Prins, H A; Cuesta, M A; Beelen, R H; Meijer, S; van Leeuwen, P A

    2000-08-01

    To analyze postoperative leukocyte functions in patients undergoing hemihepatectomy, and to assess the effect of treatment with the endotoxin-neutralizing agent bactericidal/permeability-increasing protein (rBPI21). Extensive liver resection is associated with a high incidence of infectious complications. Because elimination of pathogenic microorganisms occurs mainly by leukocytes, this increased rate of infections is most likely due to an impaired function of these cells. Endotoxin, translocated from the gut into the systemic circulation as a result of increased gut permeability and reduced hepatic clearance function after major liver resection, may play an important role in the impairment of posthepatectomy leukocyte function. To investigate whether hemihepatectomy results in impaired leukocyte functions and to determine the role of endotoxin in this process, leukocyte oxidative burst and leukocyte antigen expression were studied in three groups of patients: patients undergoing a hemihepatectomy and receiving rBPI21 treatment, patients undergoing hemihepatectomy and receiving placebo, and as an extra control group patients undergoing other major abdominal surgeries. Blood samples were collected before surgery, 2 hours after surgery, and at days 1, 2, 5, and 7. Phorbol myristate acetate-stimulated oxidative burst was measured using dihydrorhodamine, and leukocyte surface expression of the antigens CD11b, CD16, and CD14 was investigated by indirect immunofluorescence. Both oxidative burst and membrane surface expression were quantified by flow cytometry. An indication of the antiendotoxin effect of rBPI21 treatment was provided by assessment of plasma lipopolysaccharide binding protein (LBP) levels by enzyme-linked immunosorbent assay. The oxidative burst in the hemihepatectomized patients receiving placebo and the controls increased 2 hours after surgery, whereas it decreased in the rBPI21-treated patients, resulting in significant differences between the groups

  13. Increased oxidative-modifications of cytosolic proteins in 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-exposed rat liver.

    PubMed

    Upreti, Vijay V; Moon, Kwan-Hoon; Yu, Li-Rong; Lee, Insong J; Eddington, Natalie D; Ye, Xiaoying; Veenstra, Timothy D; Song, Byoung-Joon

    2011-01-01

    It is well established that 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA-exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2-DE analysis showed markedly increased levels of biotin-N-methylimide-labeled oxidized cytosolic proteins in MDMA-exposed rats compared to vehicle-treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti-oxidant defensive enzymes, a calcium-binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c-Jun N-terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti-apoptotic Bcl-2 protein, their activation may promote apoptosis in MDMA-exposed tissues. Our results show for the first time that MDMA induces oxidative-modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.

  14. Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

    PubMed

    Metcalf, Kevin J; Tullman-Ercek, Danielle

    2017-01-01

    Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

  15. HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity.

    PubMed

    Trusolino, L; Cavassa, S; Angelini, P; Andó, M; Bertotti, A; Comoglio, P M; Boccaccio, C

    2000-08-01

    Hepatocyte growth factor/scatter factor (HGF/SF) controls a genetic program known as 'invasive growth', which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth factor (EGF). Neither factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including beta1, beta3, beta4, and beta5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These data suggest that HGF/SF promotes cell adhesion and invasiveness by increasing the avidity of integrins for their specific ligands.

  16. Increased dietary protein attenuates C-reactive protein and creatine kinase responses to exercise-induced energy deficit

    USDA-ARS?s Scientific Manuscript database

    We determined if dietary protein (P) modulates responses of C-reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise-induced energy deficit (DEF). Thirteen healthy men (22 +/- 1 y, VO2peak 60 +/- 2 ml.kg-1.min-1) balanced energy expenditure (EE...

  17. Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

    PubMed Central

    Liang, Zhe; Xie, Yan; Dominguez, Jessica A.; Breed, Elise R.; Yoseph, Benyam P.; Burd, Eileen M.; Farris, Alton B.

    2014-01-01

    Background Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Methods Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. Results In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Conclusions Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice. PMID:25010671

  18. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

    PubMed Central

    Guan, J-J; Zhang, X-D; Sun, W; Qi, L; Wu, J-C; Qin, Z-H

    2015-01-01

    DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis. PMID:25633293

  19. Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes.

    PubMed

    Russo, Arianna; Scardigli, Raffaella; La Regina, Federico; Murray, Melissa E; Romano, Nicla; Dickson, Dennis W; Wolozin, Benjamin; Cattaneo, Antonino; Ceci, Marcello

    2017-04-15

    TDP-43 is a well known RNA binding protein involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia (FTLD). In physiological conditions, TDP-43 mainly localizes in the nucleus and shuttles, at least in neurons, to the cytoplasm to form TDP-43 RNA granules. In the nucleus, TDP-43 participates to the expression and splicing of RNAs, while in the cytoplasm its functions range from transport to translation of specific mRNAs. However, if loss or gain of these TDP-43 functions are affected in ALS/FTLD pathogenesis is not clear. Here, we report that TDP-43 localizes on ribosomes not only in primary neurons but also in SH-SY5Y human neuroblastoma cells. We find that binding of TDP-43 to the translational machinery is mediated by an interaction with a specific ribosomal protein, RACK1, and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect which is rescued by overexpression of RACK1. Ribosomal loss of RACK1, which excludes TDP-43 from the translational machinery, remarkably reduces formation of TDP-43 cytoplasmic inclusions in neuroblastoma cells. Finally, we corroborate the interaction between TDP-43 and RACK1 on polyribosomes of neuroblastoma cells with mis-localization of RACK1 on TDP-43 positive cytoplasmic inclusions in motor neurons of ALS patients. In conclusions, results from this study suggest that TDP-43 represents a translational repressor not only for specific mRNAs but for overall translation and that its binding to polyribosomes through RACK1 may promote, under conditions inducing ALS pathogenesis, the formation of cytoplasmic inclusions. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  1. Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown

    PubMed Central

    Cau, Laura; Pendaries, Valérie; Lhuillier, Emeline; Thompson, Paul R; Serre, Guy; Takahara, Hidenari; Méchin, Marie-Claire; Simon, Michel

    2017-01-01

    Background Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidyl-arginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. Objective To test whether external humidity plays a role, and investigate the molecular mechanisms involved. Methods Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30–50% relative humidity) were compared. Results RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. Conclusion These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation. PMID:28242341

  2. Moderate excess of dietary protein increases breast meat yield of broiler chicks.

    PubMed

    Bartov, I; Plavnik, I

    1998-05-01

    Two factorial experiments were carried out to evaluate the effects of dietary protein level, expressed as energy to protein (E:P) ratio, on yields of carcass and breast and on abdominal fat pad weight of male broiler chicks at 43 and 57 d of age. Two diets similar in their energy content and differing markedly in their protein content, in which the E:P ratios were lower and identical to the NRC (1994) recommendations, were used in Experiment 1. Four diets, the combination of two energy and two protein levels, in which the E:P ratios were lower and identical to the recommendations, were used in Experiment 2. There were no differences in feed intake or weight gain until 42 d of age between broilers fed the diets with the low and those fed the recommended E:P ratio. However, feed efficiency of the former was consistently and significantly (P < 0.05) higher. The high-energy diets did not affect feed intake but significantly improved weight gain between 7 and 28 d of age and feed efficiency until 42 d of age. In both experiments, carcass yield was not affected by dietary E:P ratio at 43 d of age; however, at 57 d of age it was increased significantly by the low E:P ratio in Experiment 1 and in birds fed the high energy diet in Experiment 2. On a factorial basis, the effect of the low E:P ratio on increasing yield was significant only in Experiment 1. The high dietary energy level increased carcass yield only at 43 d of age, but on a factorial basis its effect was significant. Carcass yield was significantly (P < 0.001) higher at 57 than at 43 d of age. In both experiments, breast meat yield was negatively correlated with dietary E:P ratio, a significant factorial effect. The difference in this variable between broilers fed the diets containing the low and the recommended E:P ratio was significant, at both ages in Experiment 1, and only at 57 d of age in Experiment 2. Breast meat yield was not affected by dietary energy level, and it was significantly (P < 0.001) higher

  3. Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers

    PubMed Central

    Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Marcusson, Jan; Brisby, Helena

    2013-01-01

    Introduction Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. Materials and Methods Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. Results CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. Conclusions Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI. PMID:24260563

  4. Aerobic Exercise Training Adaptations Are Increased by Postexercise Carbohydrate-Protein Supplementation

    PubMed Central

    Ferguson-Stegall, Lisa; McCleave, Erin; Ding, Zhenping; Doerner III, Phillip G.; Liu, Yang; Wang, Bei; Healy, Marin; Kleinert, Maximilian; Dessard, Benjamin; Lassiter, David G.; Kammer, Lynne; Ivy, John L.

    2011-01-01

    Carbohydrate-protein supplementation has been found to increase the rate of training adaptation when provided postresistance exercise. The present study compared the effects of a carbohydrate and protein supplement in the form of chocolate milk (CM), isocaloric carbohydrate (CHO), and placebo on training adaptations occurring over 4.5 weeks of aerobic exercise training. Thirty-two untrained subjects cycled 60 min/d, 5 d/wk for 4.5 wks at 75–80% of maximal oxygen consumption (VO2 max). Supplements were ingested immediately and 1 h after each exercise session. VO2 max and body composition were assessed before the start and end of training. VO2 max improvements were significantly greater in CM than CHO and placebo. Greater improvements in body composition, represented by a calculated lean and fat mass differential for whole body and trunk, were found in the CM group compared to CHO. We conclude supplementing with CM postexercise improves aerobic power and body composition more effectively than CHO alone. PMID:21773022

  5. Hunting increases phosphorylation of calcium/calmodulin-dependent protein kinase type II in adult barn owls.

    PubMed

    Nichols, Grant S; DeBello, William M

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is "off" in adults.

  6. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    PubMed Central

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults. PMID:25789177

  7. Distinct patterns of increased translocator protein in posterior cortical atrophy and amnestic Alzheimer's disease.

    PubMed

    Kreisl, William C; Lyoo, Chul Hyoung; Liow, Jeih-San; Snow, Joseph; Page, Emily; Jenko, Kimberly J; Morse, Cheryl L; Zoghbi, Sami S; Pike, Victor W; Turner, R Scott; Innis, Robert B

    2017-03-01

    We sought to determine whether patients with posterior cortical atrophy (PCA) demonstrate a pattern of binding to translocator protein 18 kDa, a marker of microglial activation, that is distinct from that in patients with amnestic presentation of Alzheimer's disease (AD). Eleven PCA patients, 11 amnestic AD patients, and 15 age-matched controls underwent positron emission tomography with (11)C-PBR28 to measure translocator protein 18 kDa. PCA patients showed greater (11)C-PBR28 binding than controls in occipital, posterior parietal, and temporal regions. In contrast, amnestic AD patients showed greater (11)C-PBR28 binding in inferior and medial temporal cortex. Increased (11)C-PBR28 binding overlapped with reduced cortical volume for both PCA and amnestic AD patients, and with areas of reduced glucose metabolism in PCA patients. While both patient groups showed diffuse amyloid binding, PCA patients showed greater binding than amnestic AD patients in bilateral occipital cortex. These results suggest that microglial activation is closely associated with neurodegeneration across different subtypes of AD. Published by Elsevier Inc.

  8. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

    PubMed

    Rood, Ilse M; Merchant, Michael L; Wilkey, Daniel W; Zhang, Terry; Zabrouskov, Vlad; van der Vlag, Johan; Dijkman, Henry B; Willemsen, Brigith K; Wetzels, Jack F; Klein, Jon B; Deegens, Jeroen K

    2015-11-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

  9. Increased reliability of nuclear magnetic resonance protein structures by consensus structure bundles.

    PubMed

    Buchner, Lena; Güntert, Peter

    2015-02-03

    Nuclear magnetic resonance (NMR) structures are represented by bundles of conformers calculated from different randomized initial structures using identical experimental input data. The spread among these conformers indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e., how close NMR conformers are to the "true" structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase precision often overestimate accuracy by tight bundles of high precision but much lower accuracy. To overcome this problem, we introduce a protocol for NMR structure determination with the software package CYANA, which produces, like the traditional method, bundles of conformers in agreement with a common set of conformational restraints but with a realistic precision that is, throughout a variety of proteins and NMR data sets, a much better estimate of structural accuracy than the precision of conventional structure bundles.

  10. Use of Methacrylic Acid-Containing Hydrogels to Increase Protein Transport Across the Intestinal Epithelium

    NASA Astrophysics Data System (ADS)

    Blanchette, James; Lopez, Jennifer; Park, Kinam; Peppas, Nicholas

    2002-03-01

    Oral protein delivery requires protection from the harsh environment of the stomach, release in the small intestine and passage from the intestinal lumen into the circulation. Hydrogels that swell in response to the pH change when passing from the stomach to the small intestine can accomplish the first two points. The ability to enhance the permeability of intestinal epithelial cells is currently under investigation. Methacrylic acid-containing hydrogels have shown the ability to bind calcium ions that decreases the concentration of free extracellular calcium for these epithelial cells. This change triggers a number of intracellular events including rearrangement of the cytoskeleton leading to increased permeability. Studies done on Caco-2 cells (human colon adenocarcinoma) measuring changes in transepithelial resistance are used to assess the effect of the polymer-cell interactions on the integrity of intestinal epithelial cell monolayers.

  11. Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

    PubMed Central

    Orsi, Andrea

    2007-01-01

    Prion protein (PrP), the causative agent of transmissible spongiform encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation. PMID:19164910

  12. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    SciTech Connect

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos; Cerpa, Waldo; Cambiazo, Veronica; Inestrosa, Nibaldo C.; Gonzalez, Mauricio

    2009-05-15

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to both Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

  13. Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.

    PubMed

    Yun, Jang-Hyuk; Park, Sung Wook; Kim, Kyung-Jin; Bae, Jong-Sup; Lee, Eun Hui; Paek, Sun Ha; Kim, Seung U; Ye, Sangkyu; Kim, Jeong-Hun; Cho, Chung-Hyun

    2017-05-01

    Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123-1134, 2017. © 2016 Wiley Periodicals, Inc.

  14. Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline.

    PubMed

    Manna, Caterina; Officioso, Arbace; Trojsi, Francesca; Tedeschi, Gioacchino; Leoncini, Silvia; Signorini, Cinzia; Ciccoli, Lucia; De Felice, Claudio

    2016-12-01

    Down syndrome (DS, trisomy 21) is the leading cause of chromosomal-related intellectual disability. At an early age, adults with DS develop with the neuropathological hallmarks of Alzheimer's disease, associated with a chronic oxidative stress. To investigate if non-protein bound iron (NPBI) can contribute to building up a pro-oxidative microenvironment, we evaluated NPBI in both plasma and erythrocytes from DS and age-matched controls, together with in vivo markers of lipid peroxidation (F2-isoprostanes, F2-dihomo-isoprostanes, F4-neuroprostanes) and in vitro reactive oxygen species (ROS) formation in erythrocytes. The serum iron panel and uric acid were also measured. Second, we explored possible correlation between NPBI, lipid peroxidation and cognitive performance. Here, we report NPBI increase in DS, which correlates with increased serum ferritin and uric acid. High levels of lipid peroxidation markers and intraerythrocyte ROS formations were also reported. Furthermore, the scores of Raven's Colored Progressive Matrices (RCPM) test, performed as a measure of current cognitive function, are inversely related to NPBI, serum uric acid, and ferritin. Likewise, ROS production, F2-isoprostanes, and F4-neuroprostanes were also inversely related to cognitive performance, whereas serum transferrin positively correlated to RCPM scores. Our data reveal that increased availability of free redox-active iron, associated with enhanced lipid peroxidation, may be involved in neurodegeneration and cognitive decline in DS. In this respect, we propose chelation therapy as a potential preventive/therapeutic tool in DS.

  15. Fear and C-reactive protein cosynergize annual pulse increases in healthy adults

    PubMed Central

    Shenhar-Tsarfaty, Shani; Yayon, Nadav; Waiskopf, Nir; Shapira, Itzhak; Toker, Sharon; Zaltser, David; Berliner, Shlomo; Ritov, Ya'acov; Soreq, Hermona

    2015-01-01

    Recent international terror outbreaks notably involve long-term mental health risks to the exposed population, but whether physical health risks are also anticipated has remained unknown. Here, we report fear of terror-induced annual increases in resting heart rate (pulse), a notable risk factor of all-cause mortality. Partial least squares analysis based on 325 measured parameters successfully predicted annual pulse increases, inverse to the expected age-related pulse decline, in approximately 4.1% of a cohort of 17,380 apparently healthy active Israeli adults. Nonbiased hierarchical regression analysis among 27 of those parameters identified pertinent fear of terror combined with the inflammatory biomarker C-reactive protein as prominent coregulators of the observed annual pulse increases. In comparison, basal pulse primarily depended on general physiological parameters and reduced cholinergic control over anxiety and inflammation, together indicating that consistent exposure to terror threats ignites fear-induced exacerbation of preexisting neuro-immune risks of all-cause mortality. PMID:25535364

  16. Increased expression of Zinc finger protein 267 in non-alcoholic fatty liver disease

    PubMed Central

    Schnabl, Bernd; Czech, Barbara; Valletta, Daniela; Weiss, Thomas S; Kirovski, Georgi; Hellerbrand, Claus

    2011-01-01

    Hepatocellular lipid accumulation is a hallmark of non-alcoholic fatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign. PMID:22076166

  17. Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

    PubMed

    Kanda, Atsushi; Nakayama, Kyosuke; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2013-09-28

    It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague–Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

  18. Interactions of promonocytic U937 cells with proteins of the extracellular matrix.

    PubMed Central

    Pucillo, C E; Colombatti, A; Vitale, M; Salzano, S; Rossi, G; Formisano, S

    1993-01-01

    Monocyte interaction with proteins of the extracellular matrix (ECM) is regulated by expression of specific cell-surface receptors. 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been shown to induce the promonocytic cell line U937 to a more differentiated monocyte-like state. In this study we have analysed the attachment of U937 cells to ECM proteins and the effects of treatment with TPA on this process. Non-induced U937 cells attach to fibronectin- and Matrigel-coated surfaces without TPA stimulation, but TPA further increases adherence to these substrates as measured by an enhanced binding and by the lower concentration of proteins needed in the substrate to achieve 50% of maximal cell adhesion. Attachment to type I collagen was seen only with activated U937 cells, whereas no measurable attachment to bovine serum albumin, vitronectin, and type IV collagen was detected. TPA-activated U937 cells showed a two-fold increase in the expression of the RGD-dependent integrin receptors alpha 3 and alpha 5, and a reduction in the expression of alpha 4, another fibronectin-specific receptor, whereas the common beta 1 chain was unchanged. Attachment of U937 cells to fibronectin was primarily mediated by the alpha 3 and alpha 5 integrins, as revealed by the ability of GRGDS peptides to inhibit attachment, whereas the CS-1 peptide, containing the alpha 4 binding site, was largely ineffective in blocking attachment. PMID:8262552

  19. Exercise but not mannitol provocation increases urinary Clara cell protein (CC16) in elite swimmers.

    PubMed

    Romberg, Kerstin; Bjermer, Leif; Tufvesson, Ellen

    2011-01-01

    Elite swimmers have an increased risk of developing asthma, and exposure to chloramine is believed to be an important trigger factor. The aim of the present study was to explore pathophysiological mechanisms behind induced bronchoconstriction in swimmers exposed to chloramine, before and after swim exercise provocation as well as mannitol provocation. Urinary Clara cell protein (CC16) was used as a possible marker for epithelial stress. 101 elite aspiring swim athletes were investigated and urinary samples were collected before and 1 h after completed exercise and mannitol challenge. CC16, 11β-prostaglandin (PG)F(2α) and leukotriene E(4) (LTE(4)) were measured. Urinary levels of CC16 were clearly increased after exercise challenge, while no reaction was seen after mannitol challenge. Similar to CC16, the level of 11β-PGF(2α) was increased after exercise challenge, but not after mannitol challenge, while LTE(4) was reduced after exercise. There was no significant difference in urinary response between those with a negative compared to positive challenge, but a tendency of increased baseline levels of 11β-PGF(2α) and LTE(4) in individuals with a positive mannitol challenge. The uniform increase of CC16 after swim exercise indicates that CC16 is of importance in epithelial stress, and may as such be an important pathogenic factor behind asthma development in swimmers. The changes seen in urinary levels of 11β-PGF(2α) and LTE(4) indicate a pathophysiological role in both mannitol and exercise challenge. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Basic science and spine literature document bone morphogenetic protein increases cancer risk.

    PubMed

    Epstein, Nancy E

    2014-01-01

    Increasingly, clinical articles document that bone morphogenetic protein (BMP/INFUSE: Medtronic, Memphis, TN, USA) and its derivatives utilized in spinal surgery increase the risk of developing cancer. However, there is also a large body of basic science articles that also document that various types of BMP and other members of the TGF-Beta (transforming growth factor beta) family promote the growth of different types of cancers. This review looks at many clinical articles citing BMP/INFUSE's role, largely "off-label", in contributing to complications encountered during spinal surgery. Next, however, specific attention is given to the clinical and basic science literature regarding how BMP and its derivatives (e.g. members of the TGF-beta family) may also impact the development of breast and other cancers. Utilizing BMP/INFUSE in spine surgery increased the risk of cancers/new malignancy as documented in several studies. For example, Carragee et al. found that for single-level instrumented posterolateral fusions (PLF) using high-dose rhBMP-2 (239 patients) vs. autograft (control group; n = 224), the risks of new cancers at 2 and 5 years postoperatively were increased. In laboratory studies, BMP's along with other members of the TGF-Beta family also modulated/contributed to the proliferation/differentiation of breast cancer (e.g. bone formation/turnover, breast cancer-related solid tumors, and metastases), lung, adrenal, and colon cancer. BMP/INFUSE when utilized clinically in spinal fusion surgery appears to promote cancer at higher rates than observed in the overall population. Furthermore, BMP and TGF-beta are correlated with increased cancer growth both in the clinic and the laboratory.

  1. Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

    PubMed Central

    Alvarez-Gallardo, Horacio; Kjelland, Michael E.; Moreno, Juan F.; Welsh, Thomas H.; Randel, Ronald D.; Lammoglia, Miguel A.; Pérez-Martínez, Mario; Lara-Sagahón, Alma V.; Esperón-Sumano, A. Enrique; Romo, Salvador

    2013-01-01

    A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented. PMID:23762288