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Sample records for ristocetin cofactor activity

  1. Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

    Science.gov (United States)

    Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

    2002-04-01

    For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

  2. Inhibition of 125I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

    International Nuclear Information System (INIS)

    Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

    1989-01-01

    Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of 125 I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids

  3. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

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

  4. Ristocetin induces phosphorylated-HSP27 (HSPB1) release from the platelets of type 2 DM patients: Anti-platelet agent-effect on the release.

    Science.gov (United States)

    Tokuda, Haruhiko; Kuroyanagi, Gen; Onuma, Takashi; Enomoto, Yukiko; Doi, Tomoaki; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2018-04-01

    It has been previously reported that HSP27 is released from platelets in type 2 diabetes mellitus (DM) patients according to phosphorylation. In the present study, we investigated the effect of ristocetin, a glycoprotein (GP)Ib/IX/V activator, on the release of HSP27 and the effect of anti-platelet agents, such as acetylsalicylic acid (ASA), on this release. Forty-six patients with type 2 DM were recruited, and classified into two groups depending on administration of anti-platelet agents, resulting in 31 patients without these agents (control group) and 15 patients with them (anti-platelet group). Ristocetin potently induced the aggregation of platelets in the two groups. Ristocetin-induced changes of the area under the curve of light transmittance and the ratio of the size of platelet aggregates in the anti-platelet group were slightly different from those in the control group. On the other hand, the levels of phosphorylated-HSP27 induced by ristocetin in the platelets from a patient of the anti-platelet group taking ASA were significantly lower than those from a patient of the control group. The levels of HSP27 release from the ristocetin-stimulated platelets were significantly correlated with the levels of phosphorylated-HSP27 in the platelets from patients in the two groups. The levels of HSP27 release and those of platelet-derived growth factor-AB (PDGF-AB) secretion stimulated by ristocetin in the anti-platelet group were lower than those in the control group. In addition, the levels of HSP27 release and those of PDGF-AB secretion stimulated by ADP in the anti-platelet group were lower than those in the control group. These results strongly suggest that anti-platelet agents inhibit the HSP27 release from platelets stimulated by ristocetin but not the aggregation in type 2 DM patients.

  5. CoFactor: Folate Requirement for Optimization of 5-Fluouracil Activity in Anticancer Chemotherapy

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    Muhammad Wasif Saif

    2010-01-01

    Full Text Available Intracellular reduced folate exists as a “pool” of more than 6 interconvertable forms. One of these forms, 5,10 methylenetetrahydrofolic acid (CH2THF, is the key one-carbon donor and reduced folate substrate for thymidylate synthase (TS. This pathway has been an important target for chemotherapy as it provides one of the necessary nucleotide substrates for DNA synthesis. The fluoropyrimidine 5-fluorouracil (5-FU exerts its main cytotoxic activity through TS inhibition. Leucovorin (5-formyltetrahydrofolate; LV has been used to increase the intracellular reduced folate pools and enhance TS inhibition. However, it must be metabolized within the cell through multiple intracellular enzymatic steps to form CH2THF. CoFactor (USAN fotrexorin calcium, (dl-5,10,-methylenepteroyl-monoglutamate calcium salt is a reduced folate that potentiates 5-FU cytotoxicity. According to early clinical trials, when 5-FU is modulated by CoFactor instead of LV, there is greater anti-tumor activity and less toxicity. This review presents the emerging role of CoFactor in colorectal and nongastrointestinal malignancies.

  6. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

    LENUS (Irish Health Repository)

    Harmon, Shona

    2008-11-07

    Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

  7. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  8. Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

    Science.gov (United States)

    Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A

    2018-04-16

    Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

  9. EFFECTS OF CIGARETTE SMOKING ON ERYTHROCYTE ANTIOXIDATIVE ENZYME ACTIVITIES AND PLASMA CONCENTRATIONS OF THEIR COFACTORS

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    M. Zahraie

    2005-07-01

    Full Text Available Tobacco smoke contains numerous compounds, many ‎of which are oxidants and capable of producing free radical and enhancing ‎the oxidative stress. The aim of this study was to investigate the effect of cigarette smoking on the erythrocyte antioxidative enzyme activities and the plasma ‎concentration of their cofactors. ‎Sixty eight healthy men were enrolled, 32 of whom had never smoked and 36 had smoked at least 10 cigarettes per day for ‎at least one year. Hemolysate superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px and ‎catalase (CAT activities were measured using spectrophotometer. Plasma copper, zinc and selenium concentrations were determined ‎using atomic absorption spectrophotometer. Plasma iron concentration was determined by colorimetric ‎method. We found that erythrocyte Cu-Zn SOD activity was significantly higher in tobacco smokers ‎compared with non-smokers (1294 ± 206.7 U/gHb in smokers vs. 1121.6 ± 237.8 U/gHb in non-‎smokers, P < 0.01. While plasma selenium concentration was significantly lower in tobacco ‎smokers (62.7±14.8 μg/L in smokers vs. 92.1 ± 17.5 μg/L in non-smokers, P < 0.01, there were no significant ‎differences in erythrocyte GSH-Px and CAT activities and plasma copper, zinc and iron concentrations between the two groups. ‎It seems that cigarette smoking can alter antioxidative enzymes activity and plasma concentration of some trace elements.

  10. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    Science.gov (United States)

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  11. Hydrogen Activation by Biomimetic [NiFe]-Hydrogenase Model Containing Protected Cyanide Cofactors

    Science.gov (United States)

    Manor, Brian C.; Rauchfuss, Thomas B.

    2013-01-01

    Described are experiments that allow incorporation of cyanide cofactors and hydride substrate into active site models [NiFe]-hydrogenases (H2ases). Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe), (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BArF3) to give the adducts (CO)2(CNBArF3)2Fe(pdt)Ni(dxpe), (1(BArF3)2, 2(BArF3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives Et4N[(CO)(CNBArF3)2Fe(H)(pdt)Ni(dxpe)], (dxpe = dppe, Et4N[H3(BArF3)2]; dxpe = dcpe, Et4N[H4(BArF3)2]). Crystallographic analysis shows that Et4N[H3(BArF3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H…Ni center is unsymmetrical with rFe-H = 1.51(3) and rNi-H = 1.71(3) Å. Both crystallographic and 19F NMR analysis show that the CNBArF3− ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BArF3)2]− and [H4(BArF3)2]− oxidize at mild potentials, near the Fc+/0 couple. Electrochemical measurements indicate that in the presence of base, [H3(BArF3)2]− catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and ammonium salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BArF3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBArF3)2Fe(pdt)(Cl)Ni(dppe)]. PMID:23899049

  12. Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights.

    Science.gov (United States)

    Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V; Kitten, Todd; Rosenzweig, Amy C; Stubbe, JoAnne

    2014-02-28

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/β2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

  13. Proteolytic activation transforms heparin cofactor II into a host defense molecule.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-06-15

    The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

  14. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity

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    Joshua R. Kane

    2015-05-01

    Full Text Available HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac] and non-primate (FIV, BIV, and MVV viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA, for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  15. Mechanism of the reaction of ebselen with endogenous thiols : dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen

    NARCIS (Netherlands)

    Haenen, G R; De Rooij, B M; Vermeulen, N P; Bast, A

    The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni

  16. Site-Specific Bioconjugation of an Organometallic Electron Mediator to an Enzyme with Retained Photocatalytic Cofactor Regenerating Capacity and Enzymatic Activity

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    Sung In Lim

    2015-04-01

    Full Text Available Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(PH being readily available to a redox enzyme, when the local NAD(PH concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH. A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  17. Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

    Science.gov (United States)

    Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

    2017-03-01

    A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Klausen, Louise Kjær; Prag, Søren

    2009-01-01

    in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26S proteasome. eEF1A1 is therefore a likely physiological substrate....... In response to knock-down of Txnl1, ubiquitin-protein conjugates were moderately stabilised. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms....

  19. Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

    Science.gov (United States)

    Kerr, Heather; Wong, Edwin; Makou, Elisavet; Yang, Yi; Marchbank, Kevin; Kavanagh, David; Richards, Anna; Herbert, Andrew P; Barlow, Paul N

    2017-08-11

    Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes ( E S ) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on E S but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of E S PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on E S Conversely, PspCN boosted the CA, on E S , of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. N1421K mutation in the glycoprotein Ib binding domain impairs ristocetin- and botrocetin-mediated binding of von Willebrand factor to platelets

    DEFF Research Database (Denmark)

    Lanke, E.; Kristoffersson, A.C.; Isaksson, C.

    2008-01-01

    , moderately decreased plasma factor VIII (FVIII) and VWF levels, and disproportionately low-plasma VWF:RCo levels. The patients were found to be heterozygous for the novel N1421K mutation, caused by a 4263C > G transversion in exon 28 of the VWF gene coding for the A1 domain. Botrocetin- and ristocetin-mediated...... binding of plasma VWF to GPIb were reduced in the patients. In vitro mutagenesis and expression in COS-7 cells confirmed the impairment of the mutant in botrocetin- and ristocetin-mediated VWF binding to GPIb. VWF collagen binding capacity was unaffected in plasma from the heterozygous individuals as well...

  1. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    International Nuclear Information System (INIS)

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  2. Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Ignatov, Artem; Miropolskaya, Nataliya; Kulbachinskiy, Andrey

    2014-10-01

    Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  4. Lanthanide Cofactors for Triphosphorylation Ribozymes

    Science.gov (United States)

    Sweeney, K. J.; Müller, U. F.

    2017-07-01

    RNA world organisms could have used trimetaphosphate as energy source for thermodynamically unfavorable RNA polymerization. Using in vitro selection we show here that Lanthanides can serve as cofactors for ribozyme-catalyzed RNA triphosphorylation.

  5. Replacing Electron Transport Cofactors with Hydrogenases

    KAUST Repository

    Laamarti, Rkia

    2016-12-01

    Enzymes have found applications in a broad range of industrial production processes. While high catalytic activity, selectivity and mild reaction conditions are attractive advantages of the biocatalysts, particularly costs arising from required cofactors pose a sever limitation. While cofactor-recycling systems are available, their use implies constraints for process set-up and conditions, which are a particular problem e.g. for solid-gas-phase reactions. Several oxidoreductases are able to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a reduction reaction circumventing redox-cofactors requirements. In such a set-up, hydrogenases could generate and provide electrons directly form gaseous hydrogen. This thesis describes the co-immobilization of the oxygen tolerant hydrogenases from C. eutropha or C. metallidurans and cytochrome P450BM3 as test system. Conductive material in the form of carbon nanotubes (CNT) serves as a suitable support. A combination of the hydrogenase and the catalytic domain of P450BM3 immobilized on carbon nanotubes were tested for the oxidation of lauric acid in the presence of hydrogen instead of an electron-transport cofactor. The GC-MS analysis reveals the conversion of 4% of lauric acid (LA) into three products, which correspond to the hydroxylated lauric acid in three different positions with a total turnover (TON) of 34. The product distribution is similar to that obtained when using the wildtype P450BM3 with the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Such electronic coupling couldn’t be achieved for the conversion of other substrates such as propane and cyclohexane, probably due to the high uncoupling rate within the heme-domain of cytochrome P450BM3 when unnatural substrates are introduced.

  6. Enzyme cofactors: Double-edged sword for catalysis

    Science.gov (United States)

    Ivanov, Ivaylo

    2013-01-01

    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  7. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers EVIDENCE FOR A DIRECT PATHWAY BETWEEN THE 4′-AMINOPYRIMIDINE N1′ ATOMS

    Energy Technology Data Exchange (ETDEWEB)

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank [Pitt; (Goettingen); (VA); (Rutgers)

    2010-11-03

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4{prime}-aminopyrimidine N1{prime} atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu{sup 571}, Glu{sup 235}, and Glu{sup 237}) and Arg{sup 606} resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. (1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. (2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu{sup 235} makes no direct contact with the cofactor. The role of the conserved Glu{sup 571} residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  8. Optimization Strategies for Hardware-Based Cofactorization

    Science.gov (United States)

    Loebenberger, Daniel; Putzka, Jens

    We use the specific structure of the inputs to the cofactorization step in the general number field sieve (GNFS) in order to optimize the runtime for the cofactorization step on a hardware cluster. An optimal distribution of bitlength-specific ECM modules is proposed and compared to existing ones. With our optimizations we obtain a speedup between 17% and 33% of the cofactorization step of the GNFS when compared to the runtime of an unoptimized cluster.

  9. Cofactor engineering for advancing chemical biotechnology.

    Science.gov (United States)

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Engineering redox balance through cofactor systems.

    Science.gov (United States)

    Chen, Xiulai; Li, Shubo; Liu, Liming

    2014-06-01

    Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Nuclear Receptor Cofactors in PPARγ-Mediated Adipogenesis and Adipocyte Energy Metabolism

    Directory of Open Access Journals (Sweden)

    Emily Powell

    2007-01-01

    Full Text Available Transcriptional cofactors are integral to the proper function and regulation of nuclear receptors. Members of the peroxisome proliferator-activated receptor (PPAR family of nuclear receptors are involved in the regulation of lipid and carbohydrate metabolism. They modulate gene transcription in response to a wide variety of ligands, a process that is mediated by transcriptional coactivators and corepressors. The mechanisms by which these cofactors mediate transcriptional regulation of nuclear receptor function are still being elucidated. The rapidly increasing array of cofactors has brought into focus the need for a clear understanding of how these cofactors interact in ligand- and cell-specific manners. This review highlights the differential effects of the assorted cofactors regulating the transcriptional action of PPARγ and summarizes the recent advances in understanding the physiological functions of corepressors and coactivators.

  12. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

    Science.gov (United States)

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-09-29

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA + ) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe , generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Co-factor engineering in lactobacilli: Effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

    DEFF Research Database (Denmark)

    Rud, Ida; Solem, Christian; Jensen, Peter Ruhdal

    2008-01-01

    The hydrolytic F-1-part of the F1F0-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity...... resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated...... to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity. (C) 2008 Elsevier Inc. All rights reserved....

  14. Programming of neurotoxic cofactor CXCL-10 in HIV-1-associated dementia: abrogation of CXCL-10-induced neuro-glial toxicity in vitro by PKC activator

    Directory of Open Access Journals (Sweden)

    Mehla Rajeev

    2012-10-01

    Full Text Available Abstract Background More than 50% of patients undergoing lifelong suppressive antiviral treatment for HIV-1 infection develop minor HIV-1-associated neurocognitive disorders. Neurological complications during HIV-1 infection are the result of direct neuronal damage by proinflammatory products released from HIV-1-infected or -uninfected activated lymphocytes, monocytes, macrophages, microglia and astrocytes. The specific pro-inflammatory products and their roles in neurotoxicity are far from clear. We investigated proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF of HIV-demented (HIV-D and HIV-nondemented (HIV-ND patients and studied their affect on neuroglial toxicity. Methods and results Bioplex array showed elevated levels of signatory chemokines or cytokines (IL-6, IFN-γ, CXCL10, MCP-1 and PDGF in the CSF of HIV-D patients (n = 7 but not in that of HIV-ND patients (n = 7. Among the signatory cytokines and chemokines, CXCL10 was distinctly upregulated in-vitro in HIV-1 (NLENG1-activated human fetal astrocytes, HIV-1 (Ba-L-infected macrophages, and HIV-1 (NLENG1-infected lymphocytes. Virus-infected macrophages also had increased levels of TNF-α. Consistently, human fetal astrocytes treated with HIV-1 and TNF-α induced the signatory molecules. CXCL10 in combination with HIV-1 synergistically enhanced neuronal toxicity and showed chemotactic activity (~ 40 fold for activated peripheral blood mononuclear cells (PBMC, suggesting the intersection of signaling events imparted by HIV-1 and CXCL10 after binding to their respective surface receptors, CXCR4 and CXCR3, on neurons. Blocking CXCR3 and its downstream MAP kinase (MAPK signaling pathway suppressed combined CXCL10 and HIV-1-induced neurotoxicity. Bryostatin, a PKC modulator and suppressor of CXCR4, conferred neuroprotection against combined insult with HIV-1 and CXCL10. Bryostatin also suppressed HIV-1 and CXCL10-induced PBMC chemotaxis. Although, therapeutic targeting

  15. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    Science.gov (United States)

    Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-04-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  16. Investigation of the cofactor controlled substrate specificity of yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Dunaway-Mariano, D.; Barry, R.J.; Brush, T.; Ting, S.J.

    1986-01-01

    The PPase reaction requires the participation of three metal ion cofactors. One metal ion binds to PP activating it for reaction and the other two bind to the enzyme activating it for catalysis. Of the metal ions tested only Mg 2+ , Zn 2+ , Co 2+ , Mn 2+ can perform all these roles. Most trivalent metal ions can function to activate the PP for reaction but cannot activate the enzyme for catalysis. The Mg 2+ activated enzyme is specific for M-PP and M-PPS complexes while the Zn 2+ activated enzyme also acts on metal complexes of PPP, PPPOR, PPOR and PPF. 18 O-Incorporation studies show that the substituted phosphoryl group of the unsymmetrical PP complexes always serves as the leaving group. To gain insight into the mechanism of the cofactor control over the substrate specificity the order of substrate/cofactor binding to the enzyme was examined. Dead end inhibition studies in which Cr(III)PP served as substrate and Mg 2+ as cofactor indicate that the mechanism is rapid equilibrium ordered (CrPP binds first) while dead end inhibitor induced activator inhibition studies with Mg 2+ and MgPP indicate that the kinetic mechanism is steady state preferred order. Cofactor-enzyme binding was studied as a function of substrate structure and the results obtained rule out interference of Mg 2+ binding by substrate analogs as an explanation for the different substrate specificities of the Zn 2+ and Mg 2+ activated enzymes

  17. Oxygen diffusion pathways in a cofactor-independent dioxygenase

    Science.gov (United States)

    Di Russo, Natali V.; Condurso, Heather L.; Li, Kunhua; Bruner, Steven D.; Roitberg, Adrian E.

    2015-01-01

    Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins. PMID:26508997

  18. On the Metal Cofactor in the Tyrosinase Family

    Directory of Open Access Journals (Sweden)

    Francisco Solano

    2018-02-01

    Full Text Available The production of pigment in mammalian melanocytes requires the contribution of at least three melanogenic enzymes, tyrosinase and two other accessory enzymes called the tyrosinase-related proteins (Trp1 and Trp2, which regulate the type and amount of melanin. The last two proteins are paralogues to tyrosinase, and they appeared late in evolution by triplication of the tyrosinase gene. Tyrosinase is a copper-enzyme, and Trp2 is a zinc-enzyme. Trp1 has been more elusive, and the direct identification of its metal cofactor has never been achieved. However, due to its enzymatic activity and similarities with tyrosinase, it has been assumed as a copper-enzyme. Recently, recombinant human tyrosinase and Trp1 have been expressed in enough amounts to achieve for the first time their crystallization. Unexpectedly, it has been found that Trp1 contains a couple of Zn(II at the active site. This review discusses data about the metal cofactor of tyrosinase and Trps. It points out differences in the studied models, and it proposes some possible points accounting for the apparent discrepancies currently appearing. Moreover, some proposals about the possible flexibility of the tyrosinase family to uptake copper or zinc are discussed.

  19. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

  20. Correction: Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity

    Directory of Open Access Journals (Sweden)

    Sugahara Kazuyuki

    2011-07-01

    Full Text Available Abstract After the publication of the work entitled "Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity", by Kozlowski et al., BMC Biochemistry 2011, 12:29, we found that the legends to Figures 2 to 5 contain serious mistakes that compromise the comprehension of the work. This correction article contains the correct text of the legends to Figures 2 to 5.

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

    Science.gov (United States)

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

    2017-08-04

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

  2. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    Science.gov (United States)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  3. Zinc is the metal cofactor of Borrelia burgdorferi peptide deformylase.

    Science.gov (United States)

    Nguyen, Kiet T; Wu, Jen-Chieh; Boylan, Julie A; Gherardini, Frank C; Pei, Dehua

    2007-12-15

    Peptide deformylase (PDF, E.C. 3.5.1.88) catalyzes the removal of N-terminal formyl groups from nascent ribosome-synthesized polypeptides. PDF contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (His, His, and Cys) and a water molecule. Previous studies revealed that the metal cofactor is a Fe2+ ion in Escherichia coli and many other bacterial PDFs. In this work, we found that PDFs from two iron-deficient bacteria, Borrelia burgdorferi and Lactobacillus plantarum, are stable and highly active under aerobic conditions. The native B. burgdorferi PDF (BbPDF) was purified 1200-fold and metal analysis revealed that it contains approximately 1.1 Zn2+ ion/polypeptide but no iron. Our studies suggest that PDF utilizes different metal ions in different organisms. These data have important implications in designing PDF inhibitors and should help address some of the unresolved issues regarding PDF structure and catalytic function.

  4. A water-forming NADH oxidase from Lactobacillus pentosus and its potential application in the regeneration of synthetic biomimetic cofactors

    Directory of Open Access Journals (Sweden)

    Claudia eNowak

    2015-09-01

    Full Text Available The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox. Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13 % FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyse the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as a by-product.

  5. RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Directory of Open Access Journals (Sweden)

    Johanna Meier-Soelch

    2018-04-01

    Full Text Available The potent proinflammatory cytokine interleukin (IL-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (shRNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

  6. Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

    Science.gov (United States)

    Senger, Moritz; Stripp, Sven T; Soboh, Basem

    2017-07-14

    Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli

    DEFF Research Database (Denmark)

    Holm, Anders Koefoed; Blank, L.M.; Oldiges, M.

    2010-01-01

    Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli...... of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli....

  8. Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase.

    Science.gov (United States)

    Li, Kunhua; Fielding, Elisha N; Condurso, Heather L; Bruner, Steven D

    2017-07-01

    The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

  9. Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF.

    Science.gov (United States)

    Li, Zhu; Kitanishi, Kenichi; Twahir, Umar T; Cracan, Valentin; Chapman, Derrell; Warncke, Kurt; Banerjee, Ruma

    2017-03-10

    IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification.

    Science.gov (United States)

    Dougherty, Edward J; Guo, Chunhua; Simons, S Stoney; Chow, Carson C

    2012-01-01

    Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available.

  11. Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo†

    Science.gov (United States)

    Cotruvo, Joseph A.; Stubbe, JoAnne

    2011-01-01

    Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (FeIII2-Y•) and dimanganese(III)-Y• (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo J.A., Jr. and Stubbe J., Biochemistry (2010) 49, 1297–1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ~95% purity, with 0.2 Y•/β2, 0.9 Mn/β2, and a specific activity of 720 nmol/min/mg. In these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo. PMID:21250660

  12. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  13. Solution Structure of LXXLL-related Cofactor Peptide of Orphan Nuclear Receptor FTZ-F1

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji Hye; Lee, Chul Jin; Jung, Jin Won; Lee, Weon Tae [Yonsei University, Seoul (Korea, Republic of)

    2012-02-15

    Functional interaction between Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product fushi tarazu (FTZ) is crucial for regulating genes related to define the identities of alternate segmental regions in the Drosophila embryo. FTZ binding to the ligand-binding domain (LBD) of FTZ-F1 is of essence in activating its transcription process. We determined solution structures of the cofactor peptide (FTZ{sup PEP}) derived from FTZ by NMR spectroscopy. The cofactor peptide showed a nascent helical conformation in aqueous solution, however, the helicity was increased in the presence of TFE. Furthermore, FTZ{sup PEP} formed α- helical conformation upon FTZ-F1 binding, which provides a receptor bound structure of FTZ{sup PEP}. The solution structure of FTZ{sup PEP} in the presence of FTZ-F1 displays a long stretch of the α-helix with a bend in the middle of helix.

  14. Solution Structure of LXXLL-related Cofactor Peptide of Orphan Nuclear Receptor FTZ-F1

    International Nuclear Information System (INIS)

    Yun, Ji Hye; Lee, Chul Jin; Jung, Jin Won; Lee, Weon Tae

    2012-01-01

    Functional interaction between Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product fushi tarazu (FTZ) is crucial for regulating genes related to define the identities of alternate segmental regions in the Drosophila embryo. FTZ binding to the ligand-binding domain (LBD) of FTZ-F1 is of essence in activating its transcription process. We determined solution structures of the cofactor peptide (FTZ PEP ) derived from FTZ by NMR spectroscopy. The cofactor peptide showed a nascent helical conformation in aqueous solution, however, the helicity was increased in the presence of TFE. Furthermore, FTZ PEP formed α- helical conformation upon FTZ-F1 binding, which provides a receptor bound structure of FTZ PEP . The solution structure of FTZ PEP in the presence of FTZ-F1 displays a long stretch of the α-helix with a bend in the middle of helix

  15. S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

    Directory of Open Access Journals (Sweden)

    Lyn L. Kailing

    2018-03-01

    Full Text Available S-adenosyl-L-homocysteine (SAH hydrolases (SAHases are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+ as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor, nor the holo-enzyme (i.e., in the NAD+-bound state were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.

  16. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Lages, Nuno; Oldiges, M.

    2009-01-01

    to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...... oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol...

  17. CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site.

    Science.gov (United States)

    Kwak, Yeonju; Schwartz, Jennifer K; Huang, Victor W; Boice, Emily; Kurtz, Donald M; Solomon, Edward I

    2015-12-01

    Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

  18. A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

    Science.gov (United States)

    Tiefenbach, Jens; Moll, Pamela R; Nelson, Meryl R; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M

    2010-03-22

    Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

  19. A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

    Directory of Open Access Journals (Sweden)

    Jens Tiefenbach

    2010-03-01

    Full Text Available Nuclear receptors (NRs belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio. The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1 respond as expected to endogenous zebrafish hormones and cofactors, 2 facilitate efficient receptor and cofactor purification, 3 respond robustly to NR hormones and drugs and 4 yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

  20. Establishment and characterization of a new and stable collagen-binding assay for the assessment of von Willebrand factor activity

    Science.gov (United States)

    Ni, Y; Nesrallah, J; Agnew, M; Geske, F J; Favaloro, E J

    2013-01-01

    Introduction Laboratory diagnosis of von Willebrand disease (VWD) requires determination of both von Willebrand factor (VWF) protein levels and activity. Current VWF activity tests include the ristocetin cofactor assay and the collagen-binding assay (VWF:CB). The goal of this investigation is to characterize a new collagen-binding assay and to determine its effectiveness in identifying VWD. Methods Analytical studies were carried out to characterize the performance of a new VWF:CB ELISA. Additionally, samples from a normal population were tested as were well-characterized type 1 and type 2 VWD samples. Results Repeatability and within-laboratory precision studies resulted in coefficients of variation (CVs) of ≤11%. A linear range of 1–354% (0.01–3.54 IU/mL) was determined, along with a limit of detection and a lower limit of quantitation of 1.6% and 4.0% (0.016 and 0.04 IU/mL), respectively. Samples tested from apparently healthy individuals resulted in a normal range of 54–217% (0.54–2.17 IU/mL). Known VWD type 1 and type 2 samples were also analyzed by the ELISA, with 99% of samples having VWF:CB below the normal reference range and an estimated 96% sensitivity and 87% specificity using a VWF collagen-binding/antigen cutoff ratio of 0.50. Conclusion This new VWF:CB ELISA provides an accurate measure of collagen-binding activity that aids in the diagnosis and differentiation of type 1 from type 2 VWD. PMID:23107512

  1. Time-resolved fluorescence analysis of the mobile flavin cofactor

    Indian Academy of Sciences (India)

    Conformational heterogeneity of the FAD cofactor in -hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations ...

  2. Van Willebrand's disease in the Western Cape

    African Journals Online (AJOL)

    agglutinate normal platelets but does not have an effect on platelets from patients with VWD. The ristocetin co-factor. (RiCo~ assay measures the ability of plasma to agglutinate fixed platelets and is therefore regarded as an indirect measure of VWF activity. Qualitative analysis of VWF antigen. 0JWF-Ag) is required for ...

  3. Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

    Science.gov (United States)

    Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

    2017-10-30

    Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D

  4. Cofactory: Sequence-based prediction of cofactor specificity of Rossmann folds

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Blom, Nikolaj; Feist, Adam

    2014-01-01

    Obtaining optimal cofactor balance to drive production is a challenge metabolically engineered microbial strains. To facilitate identification of heterologous enzymes with desirable altered cofactor requirements from native content, we have developed Cofactory, a method for prediction of enzyme...

  5. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes

    DEFF Research Database (Denmark)

    Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe

    2015-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure of Mesorhizobium loti NAT1 [(RHILO)NAT1] was determined...... for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover......, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints....

  6. Improving metabolic efficiency of the reverse beta-oxidation cycle by balancing redox cofactor requirement.

    Science.gov (United States)

    Wu, Junjun; Zhang, Xia; Zhou, Peng; Huang, Jiaying; Xia, Xiudong; Li, Wei; Zhou, Ziyu; Chen, Yue; Liu, Yinghao; Dong, Mingsheng

    2017-11-01

    Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C 6 -C 10 , MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2g/L to 3.1g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  7. Promoter and Cofactor Requirements for SERM-ER Activity

    National Research Council Canada - National Science Library

    Carroll, Jason S

    2007-01-01

    .... We originally planned to develop a methodology for specifically isolating chromatin to assess associated proteins but due to technical limitations we developed the ChiP-on-chip technique to map ER...

  8. Influence of common mucosal co-factors on HIV infection in the female genital tract.

    Science.gov (United States)

    Ferreira, Victor H; Kafka, Jessica K; Kaushic, Charu

    2014-06-01

    Women constitute almost half of HIV-infected population globally, and the female genital tract (FGT) accounts for approximately 40% of all new HIV infections worldwide. The FGT is composed of upper and lower parts, distinct in their morphological and functional characteristics. Co-factors in the genital microenvironment, such as presence of hormones, semen, and other sexually transmitted infections, can facilitate or deter HIV infection and play a critical role in determining susceptibility to HIV. In this review, we examine some of these co-factors and their potential influence. Presence of physical and chemical barriers such as epithelial tight junctions, mucus, and anti-microbial peptides can actively block and inhibit viral replication, presenting a significant deterrent to HIV. Upon exposure, HIV and other pathogens first encounter the genital epithelium: cells that express a wide repertoire of pattern recognition receptors that can recognize and directly initiate innate immune responses. These and other interactions in the genital tract can lead to direct and indirect inflammation and enhance the number of local target cells, immune activation, and microbial translocation, all of which promote HIV infection and replication. Better understanding of the dynamics of HIV transmission in the female genital tract would be invaluable for improving the design of prophylactic strategies against HIV. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. O-, N-Atoms-Coordinated Mn Cofactors within a Graphene Framework as Bioinspired Oxygen Reduction Reaction Electrocatalysts.

    Science.gov (United States)

    Yang, Yang; Mao, Kaitian; Gao, Shiqi; Huang, Hao; Xia, Guoliang; Lin, Zhiyu; Jiang, Peng; Wang, Changlai; Wang, Hui; Chen, Qianwang

    2018-05-28

    Manganese (Mn) is generally regarded as not being sufficiently active for the oxygen reduction reaction (ORR) compared to other transition metals such as Fe and Co. However, in biology, manganese-containing enzymes can catalyze oxygen-evolving reactions efficiently with a relative low onset potential. Here, atomically dispersed O and N atoms coordinated Mn active sites are incorporated within graphene frameworks to emulate both the structure and function of Mn cofactors in heme-copper oxidases superfamily. Unlike previous single-metal catalysts with general M-N-C structures, here, it is proved that a coordinated O atom can also play a significant role in tuning the intrinsic catalytic activities of transition metals. The biomimetic electrocatalyst exhibits superior performance for the ORR and zinc-air batteries under alkaline conditions, which is even better than that of commercial Pt/C. The excellent performance can be ascribed to the abundant atomically dispersed Mn cofactors in the graphene frameworks, confirmed by various characterization methods. Theoretical calculations reveal that the intrinsic catalytic activity of metal Mn can be significantly improved via changing local geometry of nearest coordinated O and N atoms. Especially, graphene frameworks containing the Mn-N 3 O 1 cofactor demonstrate the fastest ORR kinetics due to the tuning of the d electronic states to a reasonable state. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

    Science.gov (United States)

    Njuma, Olive J; Ndontsa, Elizabeth N; Goodwin, Douglas C

    2014-02-15

    Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Insights into hydrocarbon formation by nitrogenase cofactor homologs.

    Science.gov (United States)

    Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W

    2015-04-14

    The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN(-) to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN(-), which could be explained by the presence of a "free" Fe atom that is "unmasked" by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C-C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN(-) reduction without complications originating from the heterometal and homocitrate components. Nitrogenase is a metalloenzyme that is highly complex in structure and uniquely versatile in function. It catalyzes two reactions that parallel two important industrial processes: the reduction of nitrogen to ammonia, which parallels the Haber-Bosch process in ammonia production, and the reduction of carbon monoxide to hydrocarbons, which parallels the Fischer-Tropsch process in fuel production. Thus, the significance of nitrogenase can be appreciated from the perspective of the useful products it generates: (i) ammonia, the "fixed" nitrogen that is essential for the existence of the entire human population; and (ii) hydrocarbons, the "recycled" carbon fuel that could be used to directly address the worldwide energy shortage. This article provides initial insights into the catalytic characteristics of various nitrogenase cofactors in hydrocarbon formation. The reported assay system provides a useful tool for mechanistic

  12. Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

    Science.gov (United States)

    Dard, Amélie; Reboulet, Jonathan; Jia, Yunlong; Bleicher, Françoise; Duffraisse, Marilyne; Vanaker, Jean-Marc; Forcet, Christelle; Merabet, Samir

    2018-03-13

    HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lindsey N.; Koech, Phillip K.; Plymale, Andrew E.; Landorf, Elizabeth V.; Konopka, Allan; Collart, Frank; Lipton, Mary S.; Romine, Margaret F.; Wright, Aaron T.

    2016-02-02

    The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are critically needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically-predicted functions. Of particular importance are transport mechanisms, used to shuttle nutrients and metabolites across cell mem-branes, such as B vitamins, which are indispensable to metabolic reactions crucial to the survival of diverse microbes ranging from members of environmental microbial communities to human pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, and characterization of intra-cellular enzyme-cofactor/nutrient associations are needed to enable a significantly improved understanding of microbial biochemis-try and physiology, how microbes associate with others, and how they sense and respond to environmental perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular protein-cofactor associations through live cell labeling of the filamentous anoxygenic pho-toheterotroph, Chloroflexus aurantiacus J-10-fl, known for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by iden-tifying B vitamin transport and disposition mechanisms required for survival.

  14. Panning for SNuRMs: using cofactor profiling for the rational discovery of selective nuclear receptor modulators.

    Science.gov (United States)

    Kremoser, Claus; Albers, Michael; Burris, Thomas P; Deuschle, Ulrich; Koegl, Manfred

    2007-10-01

    Drugs that target nuclear receptors are clinically, as well as commercially, successful. Their widespread use, however, is limited by an inherent propensity of nuclear receptors to trigger beneficial, as well as adverse, pharmacological effects upon drug activation. Hence, selective drugs that display reduced adverse effects, such as the selective estrogen receptor modulator (SERM) Raloxifene, have been developed by guidance through classical cell culture assays and animal trials. Full agonist and selective modulator nuclear receptor drugs, in general, differ by their ability to recruit certain cofactors to the receptor protein. Hence, systematic cofactor profiling is advancing into an approach for the rationally guided identification of selective NR modulators (SNuRMs) with improved therapeutic ratio.

  15. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    Science.gov (United States)

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  16. Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

    Science.gov (United States)

    García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

    2017-08-01

    Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Roles of Fe-S proteins: from cofactor synthesis to iron homeostasis to protein synthesis.

    Science.gov (United States)

    Pain, Debkumar; Dancis, Andrew

    2016-06-01

    Fe-S cluster assembly is an essential process for all cells. Impairment of Fe-S cluster assembly creates diseases in diverse and surprising ways. In one scenario, the loss of function of lipoic acid synthase, an enzyme with Fe-S cluster cofactor in mitochondria, impairs activity of various lipoamide-dependent enzymes with drastic consequences for metabolism. In a second scenario, the heme biosynthetic pathway in red cell precursors is specifically targeted, and iron homeostasis is perturbed, but lipoic acid synthesis is unaffected. In a third scenario, tRNA modifications arising from action of the cysteine desulfurase and/or Fe-S cluster proteins are lost, which may lead to impaired protein synthesis. These defects can then result in cancer, neurologic dysfunction or type 2 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Characterization of water-forming NADH oxidases for co-factor regeneration

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; J. Charnock, Simon

    an environmentaland economic perspective [1]. Alcohol dehydrogenases (ADH) offer one such alternative. However, the reaction requires the oxidized nicotinamide co-factor (NAD+) that must be recycled due to its high cost contribution. One regeneration method that offers certain advantages is the oxidation of NADH......Traditional chemical methods for alcohol oxidation are often associated with issues such as high consumption of expensive oxidizing agents, generation of metal waste and the use of environmentally undesirable organic solvents. Developing green, selective catalysts is therefore important from...... using water forming NADH oxidases (NOX-2). The implementation of the ADH/NOX system for alcohol oxidation, however, requires consideration of several different issues. Enzyme activity and stability at relevant pH and temperature conditions, but also the tolerance to the substrates and products present...

  19. Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Rand, Kasper Dyrberg

    2014-01-01

    Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most...... inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide...... sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation...

  20. Cofactors in allergic reactions to food : physical exercise and alcohol are the most important

    NARCIS (Netherlands)

    Versluis, Astrid; van Os-Medendorp, Harmieke; Kruizinga, Astrid G; Blom, W Marty; Houben, Geert F; Knulst, André C

    2016-01-01

    INTRODUCTION: Involvement of cofactors, like physical exercise, alcohol consumption and use of several types of medication, are associated with more severe food allergic symptoms. However, there is limited evidence on how often cofactors play a role in food allergic reactions. The study aimed to get

  1. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

    2016-01-01

    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  2. Beyond the Protein Matrix : Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

    NARCIS (Netherlands)

    Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

    2013-01-01

    A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP(+) and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like

  3. Molybdenum-cofactor deficiency: an easily missed cause of neonatal convulsions

    NARCIS (Netherlands)

    Slot, H. M.; Overweg-Plandsoen, W. C.; Bakker, H. D.; Abeling, N. G.; Tamminga, P.; Barth, P. G.; van Gennip, A. H.

    1993-01-01

    Intractable seizures in the neonatal period may be caused by molybdenum-cofactor deficiency, an inborn error which combines the deficiencies of sulphite oxidase and xanthine dehydrogenase. The neurological symptoms of molybdenum cofactor and isolated sulphite oxidase deficiencies are identical. Two

  4. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R.

    2005-01-01

    TIP48 is a highly conserved eukaryotic AAA + protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis

  5. Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

    Science.gov (United States)

    Rovira, Alexander R; Fin, Andrea; Tor, Yitzhak

    2017-11-08

    The synthesis, photophysics, and biochemical utility of a fluorescent NAD + analogue based on an isothiazolo[4,3-d]pyrimidine core (N tz AD + ) are described. Enzymatic reactions, photophysically monitored in real time, show N tz AD + and N tz ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N tz AD + is converted to N tz ADH, reflecting a complementary photophysical behavior to that of the native NAD + /NADH. N tz AD + and N tz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. N tz AD + also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tz ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD + .

  6. Molybdenum cofactor deficiency: Identification of a patient with homozygote mutation in the MOCS3 gene

    NARCIS (Netherlands)

    Huijmans, Jan G. M.; Schot, Rachel; de Klerk, Johannis B. C.; Williams, Monique; de Coo, René F. M.; Duran, Marinus; Verheijen, Frans W.; van Slegtenhorst, Marjon; Mancini, Grazia M. S.

    2017-01-01

    We describe the clinical presentation and 17 years follow up of a boy, born to consanguineous parents and presenting with intellectual disability (ID), autism, "marfanoid" dysmorphic features, and moderate abnormalities of sulfite metabolism compatible with molybdenum cofactor deficiency, but normal

  7. Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins.

    Science.gov (United States)

    Ji, Hong-Fang; Chen, Lei; Zhang, Hong-Yu

    2008-08-01

    Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre-protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website. (c) 2008 Wiley Periodicals, Inc.

  8. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

    Science.gov (United States)

    Liu, Jiaheng; Li, Huiling; Zhao, Guangrong; Caiyin, Qinggele; Qiao, Jianjun

    2018-05-01

    NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

  9. Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

    Science.gov (United States)

    Breitenbach, J; Zhu, C; Sandmann, G

    2001-11-01

    Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

  10. A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

    Science.gov (United States)

    Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

    2017-02-17

    The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

  11. The glmS ribozyme cofactor is a general acid-base catalyst.

    Science.gov (United States)

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  12. Ntdin, a tobacco senescence-associated gene, is involved in molybdenum cofactor biosynthesis.

    Science.gov (United States)

    Yang, Seung Hwan; Berberich, Thomas; Miyazaki, Atsushi; Sano, Hiroshi; Kusano, Tomonobu

    2003-10-01

    To date, dozens of genes have been reported to be up-regulated with senescence in higher plants. Radish din1 and its ortholog sen1 of Arabidopsis are known as such, but their function is not clear yet. Here we have isolated their counterpart cDNA from tobacco and designated it as NTDIN: Its product, Ntdin, a 185 amino acid polypeptide with 56.8% and 54.2% identity to Atsen1 and Rsdin1, respectively, is localized in chloroplasts. Transcripts of Ntdin are induced by sulfate or nitrate but not by phosphate, suggesting its involvement in sulfur and nitrogen metabolism. A database search revealed that Ntdin shows similarity with the C-terminal region of Nicotiana plumbaginifolia Cnx5, which functions in molybdenum cofactor (Moco) biosynthesis. Transgenic tobacco plants with suppressed Ntdin are more tolerant to chlorate, a substrate analog of nitrate reductase, than controls, implying low nitrate reductase activity in the transgenic plants due to a deficiency of Moco. Indeed, enzymatic activities of two molybdoenzymes, nitrate reductase and xanthine dehydrogenase, in transgenic plants are found to be significantly lower than in control plants. Direct measurement of Moco contents reveals that those transgenic plants contain about 5% Moco of those of the control plants. Abscisic acid and indole-3-acidic acid, whose biosynthetic pathways require Moco, up-regulated Ntdin expression. Taken together, it is concluded that Ntdin functions in a certain step in Moco biosynthesis.

  13. Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

    International Nuclear Information System (INIS)

    Fanarraga, M.L.; Villegas, J.C.; Carranza, G.; Castano, R.; Zabala, J.C.

    2009-01-01

    Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes

  14. Refining the reaction mechanism of O2 towards its co-substrate in cofactor-free dioxygenases

    Directory of Open Access Journals (Sweden)

    Pedro J. Silva

    2016-12-01

    Full Text Available Cofactor-less oxygenases perform challenging catalytic reactions between singlet co-substrates and triplet oxygen, in spite of apparently violating the spin-conservation rule. In 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase, the active site has been suggested by quantum chemical computations to fine tune triplet oxygen reactivity, allowing it to interact rapidly with its singlet substrate without the need for spin inversion, and in urate oxidase the reaction is thought to proceed through electron transfer from the deprotonated substrate to an aminoacid sidechain, which then feeds the electron to the oxygen molecule. In this work, we perform additional quantum chemical computations on these two systems to elucidate several intriguing features unaddressed by previous workers. These computations establish that in both enzymes the reaction proceeds through direct electron transfer from co-substrate to O2 followed by radical recombination, instead of minimum-energy crossing points between singlet and triplet potential energy surfaces without formal electron transfer. The active site does not affect the reactivity of oxygen directly but is crucial for the generation of the deprotonated form of the co-substrates, which have redox potentials far below those of their protonated forms and therefore may transfer electrons to oxygen without sizeable thermodynamic barriers. This mechanism seems to be shared by most cofactor-less oxidases studied so far.

  15. Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB

    Directory of Open Access Journals (Sweden)

    Simon Arragain

    2017-11-01

    Full Text Available One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture, the hyperthermophile Methanocaldococcus infernus (single-domain architecture and the mesophile Klebsiella oxytoca (two-domain architecture, showing in silico characterization of their nitrogen fixation (nif gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.

  16. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    Full Text Available Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  17. Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

    Science.gov (United States)

    Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

    2012-01-01

    Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

  18. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Directory of Open Access Journals (Sweden)

    Martina Kalle

    Full Text Available Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  19. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; van der Plas, Mariena J A; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2014-01-01

    Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

  20. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors.

    Science.gov (United States)

    Montgomery, David C; Sorum, Alexander W; Guasch, Laura; Nicklaus, Marc C; Meier, Jordan L

    2015-08-20

    The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between palmitoyl coenzyme A (palmitoyl-CoA) and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular histone acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

    Science.gov (United States)

    Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

    2014-04-25

    Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

    Science.gov (United States)

    Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

    2014-09-01

    Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Expression and localization of tubulin cofactors TBCD and TBCE in human gametes.

    Science.gov (United States)

    Jiménez-Moreno, Victoria; Agirregoitia, Ekaitz

    2017-06-01

    The tubulin cofactors TBCD and TBCE play an essential role in regulation of the microtubule dynamics in a wide variety of somatic cells, but little information is known about the expression of these cofactors in human sperm and oocytes. In this study, we focused on the investigation of the presence of, and the differential distribution of, the tubulin cofactors TBCD and TBCE in human sperm and during human oocyte maturation. We performed expression assays for TBCD and TBCE by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescence and verified the presence of both cofactors in human gametes. TBCD and TBCE were located mainly in the middle region and in the tail of the sperm while in the oocyte the localization was cytosolic. The mRNA of both tubulin cofactors were present in the human oocytes but not in sperm cells. This finding gives a first insight into where TBCD and TBCE could carry out their function in the continuous changes that the cytoskeleton experiences during gametogenesis and also prior to fertilization.

  4. Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

    Science.gov (United States)

    Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

    2013-11-01

    The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

  5. Acute TNF-induced repression of cell identity genes is mediated by NFκB-directed redistribution of cofactors from super-enhancers

    DEFF Research Database (Denmark)

    Schmidt, Søren Fisker; Larsen, Bjørk Ditlev; Loft, Anne

    2015-01-01

    The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the...... specifically repressing super-enhancer-associated cell identity genes....... binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show...... that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby...

  6. Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase.

    Science.gov (United States)

    Pang, Jiayun; Scrutton, Nigel S; Sutcliffe, Michael J

    2014-09-01

    A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation.

    Directory of Open Access Journals (Sweden)

    Sven T Stripp

    Full Text Available [NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S rather than thiocyanate (-S-C≡N. This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation.

  8. Crystal Structure of the Thermus thermophilus 16 S rRNA Methyltransferase RsmC in Complex with Cofactor and Substrate Guanosine

    Energy Technology Data Exchange (ETDEWEB)

    Demirci, H.; Gregory, S; Dahlberg, A; Jogl, G

    2008-01-01

    Post-transcriptional modification is a ubiquitous feature of ribosomal RNA in all kingdoms of life. Modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. Here we describe high resolution crystal structures for the N{sup 2}-guanine methyltransferase RsmC that modifies residue G1207 in 16 S rRNA near the decoding site of the 30 S ribosomal subunit. RsmC is a class I S-adenosyl-l-methionine-dependent methyltransferase composed of two methyltransferase domains. However, only one S-adenosyl-l-methionine molecule and one substrate molecule, guanosine, bind in the ternary complex. The N-terminal domain does not bind any cofactor. Two structures with bound S-adenosyl-l-methionine and S-adenosyl-l-homocysteine confirm that the cofactor binding mode is highly similar to other class I methyltransferases. Secondary structure elements of the N-terminal domain contribute to cofactor-binding interactions and restrict access to the cofactor-binding site. The orientation of guanosine in the active site reveals that G1207 has to disengage from its Watson-Crick base pairing interaction with C1051 in the 16 S rRNA and flip out into the active site prior to its modification. Inspection of the 30 S crystal structure indicates that access to G1207 by RsmC is incompatible with the native subunit structure, consistent with previous suggestions that this enzyme recognizes a subunit assembly intermediate.

  9. [On the influence of local molecular environment on the redox potential of electron transfer cofactors in bacterial photosynthetic reaction centers].

    Science.gov (United States)

    Krasil'nikov, P M; Noks, P P; Rubin, A B

    2011-01-01

    The addition of cryosolvents (glycerol, dimethylsulfoxide) to a water solution containing bacterial photosynthetic reaction centers changes the redox potential of the bacteriochlorophyll dimer, but does not affect the redox potential of the quinone primary acceptor. It has been shown that the change in redox potential can be produced by changes of the electrostatic interactions between cofactors and the local molecular environment modified by additives entered into the solution. The degree of influence of a solvent on the redox potential of various cofactors is determined by degree of availability of these cofactors for molecules of solvent, which depends on the arrangement of cofactors in the structure of reaction centers.

  10. Separation of xylose and glucose using an integrated membrane system for enzymatic cofactor regeneration and downstream purification

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Sigurdardóttir, Sigyn Björk; Meyer, Anne S.

    2017-01-01

    Mixtures of xylose, glucose and pyruvate were fed to a membrane bioreactor equipped with a charged NF membrane (NTR 7450). Value-added products were obtained in the reactor via enzymatic cofactor-dependent catalysis of glucose to gluconic acid and pyruvate to lactic acid, respectively. The initial...... cofactor (NADH) concentration could be decreased to 10% of the stoichiometric value (relative to glucose) without compromising process time and substrate conversion via i) efficient cofactor regeneration and ii) high retention of cofactor (R=0.98) in the membrane bioreactor. Furthermore, accumulation...

  11. The Antioxidant Cofactor Alpha-Lipoic Acid May Control Endogenous Formaldehyde Metabolism in Mammals

    Directory of Open Access Journals (Sweden)

    Anastasia V. Shindyapina

    2017-12-01

    Full Text Available The healthy human body contains small amounts of metabolic formaldehyde (FA that mainly results from methanol oxidation by pectin methylesterase, which is active in a vegetable diet and in the gastrointestinal microbiome. With age, the ability to maintain a low level of FA decreases, which increases the risk of Alzheimer's disease and dementia. It has been shown that 1,2-dithiolane-3-pentanoic acid or alpha lipoic acid (ALA, a naturally occurring dithiol and antioxidant cofactor of mitochondrial α-ketoacid dehydrogenases, increases glutathione (GSH content and FA metabolism by mitochondrial aldehyde dehydrogenase 2 (ALDH2 thus manifests a therapeutic potential beyond its antioxidant property. We suggested that ALA can contribute to a decrease in the FA content of mammals by acting on ALDH2 expression. To test this assumption, we administered ALA in mice in order to examine the effect on FA metabolism and collected blood samples for the measurement of FA. Our data revealed that ALA efficiently eliminated FA in mice. Without affecting the specific activity of FA-metabolizing enzymes (ADH1, ALDH2, and ADH5, ALA increased the GSH content in the brain and up-regulated the expression of the FA-metabolizing ALDH2 gene in the brain, particularly in the hippocampus, but did not impact its expression in the liver in vivo or in rat liver isolated from the rest of the body. After ALA administration in mice and in accordance with the increased content of brain ALDH2 mRNA, we detected increased ALDH2 activity in brain homogenates. We hypothesized that the beneficial effects of ALA on patients with Alzheimer's disease may be associated with accelerated ALDH2-mediated FA detoxification and clearance.

  12. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    Science.gov (United States)

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

    Science.gov (United States)

    Prier, Christopher K; Arnold, Frances H

    2015-11-11

    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  14. Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus.

    Directory of Open Access Journals (Sweden)

    Nastassia Havarushka

    Full Text Available Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 °C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 °C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.

  15. Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

    Science.gov (United States)

    Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro

    2017-01-01

    Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

  16. Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori.

    Science.gov (United States)

    Fujii, Tsuguru; Yamamoto, Kimiko; Banno, Yutaka

    2016-06-01

    Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. The crystal structure of escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis

    International Nuclear Information System (INIS)

    Sanishvili, R.; Beasley, S.; Skarina, T; Glesne, D; Joachimiak, A; Edwards, A; Savchenko, A.; Univ. Health Network; Univ. of Toronto

    2004-01-01

    The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6 Angstrom resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. Coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis which can be tested in further studies

  18. Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

    Science.gov (United States)

    Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

    2014-06-04

    In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

  19. Engineering cofactor flexibility enhanced 2,3-butanediol production in Escherichia coli.

    Science.gov (United States)

    Liang, Keming; Shen, Claire R

    2017-12-01

    Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions.

  20. Reorientational properties of fluorescent analogues of the protein kinase C cofactors diacylglycerol and phorbol ester.

    NARCIS (Netherlands)

    Pap, E.H.W.; Ketelaars, M.; Borst, J.W.; Hoek, van A.; Visser, A.J.W.G.

    1996-01-01

    The reorientational properties of the fluorescently labelled protein kinase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolved polarised fluorescence. The sn-2 acyl chain of DG was replaced by diphenylhexatriene-

  1. Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

    Science.gov (United States)

    Singh, Shalini; Papareddy, Praveen; Kalle, Martina; Schmidtchen, Artur; Malmsten, Martin

    2013-11-01

    Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. Copyright © 2013. Published by Elsevier B.V.

  2. Investigation of glycerol assimilation and cofactor metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Holm, Anders Koefoed

    of glycerol kinase from L. lactis, introduction of a heterologous glycerol assimilation pathway and construction of a library of NADH oxidase activity. Based on a preliminary analysis of transcription level data, an attempt was made to stimulate glycerol assimilation by overexpressing the glycerol kinase...... already present in L. lactis. The construction and verification of a strain with increased glycerol kinase activity was not fully completed and is still ongoing. Similarly the construction of mutants expressing a heterologous pathway for glycerol dissimilation is also an ongoing task. An artificial...... effects and improve the growth rate, though not completely to the level of the reference strain. The fact that this effect was predominantly observed while utilizing xylose implicates the involvement of the pentose phosphate pathway. A possible mechanism underlying the observed growth characteristics...

  3. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

    Science.gov (United States)

    Sorice, M; Pittoni, V; Griggi, T; Losardo, A; Leri, O; Magno, M S; Misasi, R; Valesini, G

    2000-01-01

    The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β2-glycoprotein I (β2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins. PMID:10792380

  4. Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor.

    Directory of Open Access Journals (Sweden)

    Tatsunari Kondoh

    Full Text Available It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35 is found in the little brown bat (Myotis lucifugus genome. Putative mlEFL35-derived protein (mlEFL35p contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor.

  5. Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

    Science.gov (United States)

    Soutter, Holly H; Centrella, Paolo; Clark, Matthew A; Cuozzo, John W; Dumelin, Christoph E; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Kennedy, Kaitlyn M; Sigel, Eric A; Troast, Dawn M; Zhang, Ying; Ferguson, Andrew D; Davies, Gareth; Stead, Eleanor R; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A

    2016-12-06

    Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (10 11 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

  6. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

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

  7. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    Science.gov (United States)

    Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

    2018-01-01

    Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which

  8. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    Directory of Open Access Journals (Sweden)

    Zvonimir Marelja

    2018-02-01

    Full Text Available Iron sulfur (Fe-S clusters and the molybdenum cofactor (Moco are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii increased iron transiently displaces manganese on superoxide

  9. Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    King, Zachary A.; Feist, Adam

    2014-01-01

    Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity "swaps" for oxidoreductase enzymes...... specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast-including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine-and non-native products in E. coli-including 1...

  10. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

    Science.gov (United States)

    Cruz-Reyes, Jorge; Mooers, Blaine H.M.; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    2016-01-01

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. PMID:27540585

  11. Kinetics based reaction optimization of enzyme catalysed reduction of formaldehyde to methanol with synchronous cofactor regeneration

    DEFF Research Database (Denmark)

    Marpani, Fauziah Binti; Sárossy, Zsuzsa; Pinelo, Manuel

    2017-01-01

    regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor...... regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization...... experiments were conducted to verify the kinetically modelled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes...

  12. A toxic imbalance of Hsp70s in Saccharomyces cerevisiae is caused by competition for cofactors.

    Science.gov (United States)

    Keefer, Kathryn M; True, Heather L

    2017-09-01

    Molecular chaperones are responsible for managing protein folding from translation through degradation. These crucial machines ensure that protein homeostasis is optimally maintained for cell health. However, 'too much of a good thing' can be deadly, and the excess of chaperones can be toxic under certain cellular conditions. For example, overexpression of Ssa1, a yeast Hsp70, is toxic to cells in folding-challenged states such as [PSI+]. We discovered that overexpression of the nucleotide exchange factor Sse1 can partially alleviate this toxicity. We further argue that the basis of the toxicity is related to the availability of Hsp70 cofactors, such as Hsp40 J-proteins and nucleotide exchange factors. Ultimately, our work informs future studies about functional chaperone balance and cautions against therapeutic chaperone modifications without a thorough examination of cofactor relationships. © 2017 John Wiley & Sons Ltd.

  13. Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Lu, Lu; Nan, Jie; Mi, Wei; Wei, Chun-Hong; Li, Lan-Fen; Li, Yi

    2010-01-01

    Tubulin-folding cofactor A from A. thaliana has been crystallized and preliminarily analyzed using X-ray diffraction. Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the β-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289 K. The crystal diffracted to 1.6 Å resolution using synchrotron radiation and belonged to space group I4 1 , with unit-cell parameters a = 55.0, b = 55.0, c = 67.4 Å

  14. Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration.

    Science.gov (United States)

    Marpani, Fauziah; Sárossy, Zsuzsa; Pinelo, Manuel; Meyer, Anne S

    2017-12-01

    Enzymatic reduction of carbon dioxide (CO 2 ) to methanol (CH 3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH 3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO 2 to CH 3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH 3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH 3 OH, a TTN of 160 and BPR of 24 μmol CH 3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH 3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency. © 2017 Wiley Periodicals, Inc.

  15. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor.

    Science.gov (United States)

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven

    2011-08-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

  16. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    OpenAIRE

    Montgomery, David C.; Sorum, Alexander W.; Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the met...

  17. Quantum localization and protein-assisted vibrational energy flow in cofactors

    International Nuclear Information System (INIS)

    Leitner, David M

    2010-01-01

    Quantum effects influence vibrational dynamics and energy flow in biomolecules, which play a central role in biomolecule function, including control of reaction kinetics. Lifetimes of many vibrational modes of proteins and their temperature dependence, as determined by quantum golden-rule-based calculations, exhibit trends consistent with experimental observation and distinct from estimates based on classical modeling. Particularly notable are quantum coherence effects that give rise to localization of vibrational states of sizable organic molecules in the gas phase. Even when such a molecule, for instance a cofactor, is embedded in a protein, remnants of quantum localization survive that influence vibrational energy flow and its dependence on temperature. We discuss these effects on the mode-damping rates of a cofactor embedded in a protein, using the green fluorescent protein chromophore as a specific example. We find that for cofactors of this size embedded in their protein and solvent environment at room temperature a golden-rule calculation often overestimates the mode-damping rate.

  18. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

    International Nuclear Information System (INIS)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2006-01-01

    The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P2 1 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively. The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2 1 , with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2 1 and R32, respectively

  19. Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

    Science.gov (United States)

    Li, Yajun; Zhang, Jiachang; Zhang, Juan; Hao, Ling; Hua, Jinping; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-08-01

    LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

    Science.gov (United States)

    Hayward, C P M; Moffat, K A; Graf, L

    2014-06-01

    Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays. © 2014 John Wiley & Sons Ltd.

  1. Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A : a prospective cohort study

    NARCIS (Netherlands)

    Schwahn, Bernd C.; Van Spronsen, Francjan J.; Belaidi, Abdel A.; Bowhay, Stephen; Christodoulou, John; Derks, Terry G.; Hennermann, Julia B.; Jameson, Elisabeth; Koenig, Kai; McGregor, Tracy L.; Font-Montgomery, Esperanza; Santamaria-Araujo, Jose A.; Santra, Saikat; Vaidya, Mamta; Vierzig, Anne; Wassmer, Evangeline; Weis, Ilona; Wong, Flora Y.; Veldman, Alex; Schwarz, Guenter

    2015-01-01

    Background Molybdenum cofactor deficiency (MoCD) is characterised by early, rapidly progressive postnatal encephalopathy and intractable seizures, leading to severe disability and early death. Previous treatment attempts have been unsuccessful. After a pioneering single treatment we now report the

  2. De Novo Construction of Redox Active Proteins.

    Science.gov (United States)

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. © 2016 Elsevier Inc. All rights reserved.

  3. Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease.

    Science.gov (United States)

    Trotta, Lucia; Weigt, Kathleen; Schinnerling, Katina; Geelhaar-Karsch, Anika; Oelkers, Gerrit; Biagi, Federico; Corazza, Gino Roberto; Allers, Kristina; Schneider, Thomas; Erben, Ulrike; Moos, Verena

    2017-08-01

    Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei , the proportions of activated effector CD4 + T cells, determined as CD40L + IFN-γ + , were significantly lower in patients with CWD than in healthy controls; CD8 + T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei -specific degranulation, although CD69 + IFN-γ + CD8 + T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei -derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei -derived proteins may contribute to the pathogenesis of CWD. Copyright © 2017 American Society for Microbiology.

  4. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Hoff, Tine; Frandsen, Gitte Inselmann; Rocher, Anne

    1998-01-01

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde...... oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis....

  5. Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase

    International Nuclear Information System (INIS)

    Champness, J.N.; Stammers, D.K.; Beddell, C.R.

    1986-01-01

    The structure of the complex between E. coli form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. The authors discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme. (Auth.)

  6. Markers, Cofactors and Staging Systems in the Study of HIV Disease Progression: A Review

    Directory of Open Access Journals (Sweden)

    MC Portela

    1997-07-01

    Full Text Available This paper is aimed at providing a comprehensive review of markers, cofactors and staging systems used for HIV disease, focusing on some aspects that nowadays could even be considered historical, and advancing in current issues such as the prognostic value of viral load measurements, viral genotypic and phenotypic characterization, and new HIV disease treatment protocols. CD4+ cell values, combined with the new viral markers mentioned are promising as a parsimonious predictor set for defining both severity and progression. An adequate predictor of patient resource use for planning purposes still needs to be defined

  7. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    Science.gov (United States)

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  8. Cervical carcinogenesis: the role of co-factors and generation of reactive oxygen species Carcinogénesis cervical: co-factores y antioxidantes

    Directory of Open Access Journals (Sweden)

    Anna Giuliano

    2003-01-01

    Full Text Available Several HPV co-factors have been proposed, some more or less consistently associated with cervical dysplasia and cancer risk. More research, using prospective cohort designs, is needed to further describe where in carcinogenesis these factors are working and to assess the biological mechanism of these factors. In addition, further research is needed to define the role of various hormonal contraceptive formulations in promoting cervical carcinogenesis. While many interesting scientific questions remain to be answered, results from the numerous epidemiological studies conducted to date indicate that cervical dysplasia and cancer may be reduced if the oxidant antioxidant ratio is shifted to more of and antioxidant profile. In addition to cervical cancer screening, a reduction in cervical cancer incidence may be accomplished by reducing tobacco use, increasing nutritional status, and utilizing barrier contraception to prevent infection with other sexually acquired infections.Diversos co-factores de riesgo han sido asociados consistentemente con displasia cervical y cáncer invasor. Es necesario un mayor número de investigaciones que utilicen diseños de cohorte prospectivos para describir el proceso de carcinogénesis y el mecanismo biológico de cada uno de estos factores. Adicionalmente, futuras investigaciones serán necesarias para definir el papel de los anticonceptivos hormonales en la promoción de la carcinogénesis cervical. Mientras que muchas preguntas científicas interesantes permanecen sin ser respondidas, resultados de numerosos estudios epidemiológicos que se desarrollan actualmente, indican que la displasia cervical y cáncer podrán ser reducidos si la tasa de oxidantes-antioxidantes es cambiada a más de un perfil antioxidante. Además de la detección oportuna de cáncer cervical, puede lograrse una reducción de la incidencia de esta enfermedad disminuyendo el consumo de tabaco, incrementando el estatus nutricional, y

  9. Interaction with the Redox Cofactor MYW and Functional Role of a Mobile Arginine in Eukaryotic Catalase-Peroxidase

    Science.gov (United States)

    2016-01-01

    Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases with an additional posttranslationally formed redox-active Met-Tyr-Trp cofactor that is essential for catalase activity. On the basis of studies of bacterial KatGs, controversial mechanisms of hydrogen peroxide oxidation were proposed. The recent discovery of eukaryotic KatGs with differing pH optima of catalase activity now allows us to scrutinize those postulated reaction mechanisms. In our study, secreted KatG from the fungus Magnaporthe grisea (MagKatG2) was used to analyze the role of a remote KatG-typical mobile arginine that was shown to interact with the Met-Tyr-Trp adduct in a pH-dependent manner in bacterial KatGs. Here we present crystal structures of MagKatG2 at pH 3.0, 5.5, and 7.0 and investigate the mobility of Arg461 by molecular dynamics simulation. Data suggest that at pH ≥4.5 Arg461 mostly interacts with the deprotonated adduct Tyr. Elimination of Arg461 by mutation to Ala slightly increases the thermal stability but does not alter the active site architecture or the kinetics of cyanide binding. However, the variant Arg461Ala lost the wild-type-typical optimum of catalase activity at pH 5.25 (kcat = 6450 s–1) but exhibits a broad plateau between pH 4.5 and 7.5 (kcat = 270 s–1 at pH 5.5). Moreover, significant differences in the kinetics of interconversion of redox intermediates of wild-type and mutant protein mixed with either peroxyacetic acid or hydrogen peroxide are observed. These findings together with published data from bacterial KatGs allow us to propose a role of Arg461 in the H2O2 oxidation reaction of KatG. PMID:27293030

  10. Cofactor balance by nicotinamide nucleotide transhydrogenase (NNT) coordinates reductive carboxylation and glucose catabolism in the tricarboxylic acid (TCA) cycle.

    Science.gov (United States)

    Gameiro, Paulo A; Laviolette, Laura A; Kelleher, Joanne K; Iliopoulos, Othon; Stephanopoulos, Gregory

    2013-05-03

    Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)(+) cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)(+) ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.

  11. Cooperation of decay-accelerating factor and membrane cofactor protein in regulating survival of human cervical cancer cells

    International Nuclear Information System (INIS)

    Gao, Ling-Juan; Guo, Shu-Yu; Cai, You-Qun; Gu, Ping-Qing; Su, Ya-Juan; Gong, Hui; Liu, Yun; Chen, Chen

    2009-01-01

    Decay-accelerating factor (DAF) and membrane cofactor protein (MCP) are the key molecules involved in cell protection against autologus complement, which restricts the action of complement at critical stages of the cascade reaction. The cooperative effect of DAF and MCP on the survival of human cervical cancer cell (ME180) has not been demonstrated. In this study we applied, for the first time, short hairpin RNA (shRNA) to knock down the expression of the DAF and MCP with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the cooperative effects of DAF and MCP on the viability and migration, moreover the proliferation of ME180 cell. The results showed that shRNA inhibition of DAF and MCP expression enhanced complement-dependent cytolysis (CDC) up to 39% for MCP and up to 36% for DAF, and the combined inhibition of both regulators yielded further additive effects in ME180 cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two protein individually. These data indicated that combined DAF and MCP shRNA described in this study may offer an additional alternative to improve the efficacy of antibody-and complement-based cancer immunotherapy

  12. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    International Nuclear Information System (INIS)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  13. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  14. Tentative characterization of precursor compounds and co-factors of pigment formation in production of 'wu mi' from Vaccinium bracteatum Thunb. Leaves.

    Science.gov (United States)

    Fan, Mingcong; Fan, Yihui; Huang, Weiping; Wang, Li; Li, Yan; Qian, Haifeng; Zhang, Hui; Qi, Xiguang

    2018-10-01

    Vaccinium bracteatum leaves (VBTL) are traditionally used in China to dye rice grains, which assume a deep blue color, named 'Wu mi'. Information on the mechanism of pigment formation is limited. In this study, CIELAB color space parameters were used to represent the color of 'Wu mi'. Precursor compounds of pigments formed during the dyeing process were identified by UPLC Q-TOF MS analysis. The changes in co-factors for pigment formation in VBTL were measured at different growth stages. The L ∗ and b ∗ values of dyed rice increased as the leaves aged, whereas a ∗ values showed irregular changes. Six compounds were tentatively identified as pigment precursors by UPLC Q-TOF MS analysis. The pH and β-glucosidase activity at different growth stages of VBTL were indicated to be crucial co-factors for pigment formation. A tentative hypothesis is presented that iridoid glycosides are hydrolyzed by acids and β-glucosidases to form a dialdehyde structure that binds covalently with amino residues of lysine side chains in rice protein molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2017-03-17

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. How Diverse are the Protein-Bound Conformations of Small-Molecule Drugs and Cofactors?

    Science.gov (United States)

    Friedrich, Nils-Ole; Simsir, Méliné; Kirchmair, Johannes

    2018-03-01

    Knowledge of the bioactive conformations of small molecules or the ability to predict them with theoretical methods is of key importance to the design of bioactive compounds such as drugs, agrochemicals and cosmetics. Using an elaborate cheminformatics pipeline, which also evaluates the support of individual atom coordinates by the measured electron density, we compiled a complete set (“Sperrylite Dataset”) of high-quality structures of protein-bound ligand conformations from the PDB. The Sperrylite Dataset consists of a total of 10,936 high-quality structures of 4548 unique ligands. Based on this dataset, we assessed the variability of the bioactive conformations of 91 small molecules—each represented by a minimum of ten structures—and found it to be largely independent of the number of rotatable bonds. Sixty-nine molecules had at least two distinct conformations (defined by an RMSD greater than 1 Å). For a representative subset of 17 approved drugs and cofactors we observed a clear trend for the formation of few clusters of highly similar conformers. Even for proteins that share a very low sequence identity, ligands were regularly found to adopt similar conformations. For cofactors, a clear trend for extended conformations was measured, although in few cases also coiled conformers were observed. The Sperrylite Dataset is available for download from http://www.zbh.uni-hamburg.de/sperrylite_dataset.

  17. Neutrino mass matrices with two vanishing cofactors and Fritzsch texture for charged lepton mass matrix

    Science.gov (United States)

    Wang, Weijian; Guo, Shu-Yuan; Wang, Zhi-Gang

    2016-04-01

    In this paper, we study the cofactor 2 zero neutrino mass matrices with the Fritzsch-type structure in charged lepton mass matrix (CLMM). In the numerical analysis, we perform a scan over the parameter space of all the 15 possible patterns to get a large sample of viable scattering points. Among the 15 possible patterns, three of them can accommodate the latest lepton mixing and neutrino mass data. We compare the predictions of the allowed patterns with their counterparts with diagonal CLMM. In this case, the severe cosmology bound on the neutrino mass set a strong constraint on the parameter space, rendering two patterns only marginally allowed. The Fritzsch-type CLMM will have impact on the viable parameter space and give rise to different phenomenological predictions. Each allowed pattern predicts the strong correlations between physical variables, which is essential for model selection and can be probed in future experiments. It is found that under the no-diagonal CLMM, the cofactor zeros structure in neutrino mass matrix is unstable as the running of renormalization group (RG) from seesaw scale to the electroweak scale. A way out of the problem is to propose the flavor symmetry under the models with a TeV seesaw scale. The inverse seesaw model and a loop-induced model are given as two examples.

  18. A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

    International Nuclear Information System (INIS)

    Fang Jianhua; Kubota, Satoshi; Yang Bin; Zhou Naiming; Zhang Hui; Godbout, Roseline; Pomerantz, Roger J.

    2004-01-01

    HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as 'bait'. DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics

  19. Antimutagenic activity of oxidase enzymes

    International Nuclear Information System (INIS)

    Agabeili, R.A.

    1986-01-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity

  20. Cofactor interactions in the activation of tissue non-specific alkaline ...

    African Journals Online (AJOL)

    JTEkanem

    stabilization of protein structure and regulation of enzymatic ... structure of ALP at low and high concentrations respectively4,5. ..... Zhang, Y.X., Zhu, Y., Xi, H.W., Liu, Y.L. and Zhou, H.M. ... Garen, A. and Levinthal, C. (1960) A fine structure ...

  1. Quantification of methanogenic biomass by enzyme-linked immunosorbent assay and by analysis of specific methanogenic cofactors

    Energy Technology Data Exchange (ETDEWEB)

    Gorris, L G.M.; Kemp, H A; Archer, D B

    1987-01-01

    The reliability and accuracy with which enzyme-linked immunosorbent assay (ELISA) and an assay of methanogenic cofactors detect and quantify methanogenic species were investigated. Both assays required standardization with laboratory cultures of methanogenic bacteria and were applied to mixtures of pure cultures and samples from anaerobic digesters. ELISA was shown to be a simple method for detecting and quantifying individual methanogenic species. The range of species which can be assayed is limited by the range of antisera available but, potentially, ELISA can be applied to all methanogens. Although the cofactor assay is not species-specific it can distinguish hydrogenotrophic and acetotrophic methanogens and is quantitative.

  2. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

    Science.gov (United States)

    Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

    2018-05-01

    In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

  3. Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

    Science.gov (United States)

    Brown, Kyle L; Hudson, Billy G; Voziyan, Paul A

    2018-05-01

    The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered. Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell. Moreover, bromide is an essential cofactor in enzymatic cross-linking that reinforces the stability of scaffolds. Halogenation and halogen-induced oxidation of the collagen IV scaffold in disease states damage scaffold function. Halogens play an essential role in the formation of collagen IV scaffolds of basement membranes. Pathogenic damage of these scaffolds by halogenation and halogen-induced oxidation is a potential target for therapeutic interventions.

  4. Characterization of a "TRAMP-like" co-factor of the human RNA exosome

    DEFF Research Database (Denmark)

    Christensen, Marianne Skovgaard; Kristiansen, Maiken Søndergaard; Lubas, Michal Szymon

    Genome-wide studies in yeast, plants and humans have revealed numerous new transcripts in what was previously thought to be silent DNA or junk DNA. One class of non-coding transcript discovered recently is the PROMoter uPstream Transcripts (PROMPTs), which is only seen upon depletion of the RNA...... exosome, the major 3’-5’ exonuclease complex in human cells. PROMPTs have a lot in common with the yeast Cryptic Unstable Transcripts (CUTs), which are degraded by the concerted effort of the exosome, and its co-factor complex TRAMP (Trf4p/Air1p/Mtr4p). We have identified human proteins with functional...... similarities to components of the yeast TRAMP complex, and show that these are involved in the degradation of PROMPTs. While, these proteins form transient complexes with the exosome, our preliminary results also indicate that complex formation can occur directly with catalytic components of the exosome...

  5. Identification and Characterization of the Novel p97 co-factors, Rep8 and ASPL

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær

    to the ER membrane with the UBX domain situated in the cytosol. Mouse Rep8 is highly tissue-specific and abundant in gonads. In tests, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Additional precipitation experiments revealed that Rep8......The highly conserved and ubiquitin-specific AAA ATPase p97 acts on ubiquitylated substrates in diverse cellular mechanisms such as chromatin-associated degradation, fusion of homotypic membranes and ER-associated degradation. Different p97 cofactors associate with the ATPase, thereby constituting...... that ASPL localizes to the ER membrane and in vitro ASPL leads to disassembly of the p97 hexameric ATPase. Rep8 was found to interact with p97 both in vitro and in vivo, and the binding was mediated through the N-domain of p97 and the UBX domain of Rep8. Localization studies showed that Rep8 localizes...

  6. Income poverty, poverty co-factors, and the adjustment of children in elementary school.

    Science.gov (United States)

    Ackerman, Brian P; Brown, Eleanor D

    2006-01-01

    Since 1990, there have been great advances in how developmental researchers construct poverty. These advances are important because they may help inform social policy at many levels and help frame how American culture constructs poverty for children, both symbolically and in the opportunities children and families get to escape from poverty. Historically, developmental perspectives have embodied social address and main effects models, snapshot views of poverty effects at single points in time, and a rather narrow focus on income as the symbolic marker of the ecology of disadvantage. More recent views, in contrast, emphasize the diverse circumstances of disadvantaged families and diverse outcomes of disadvantaged children, the multiple sources of risk and the multiple determinants of poor outcomes for these children, dynamic aspects of that ecology, and change as well as continuity in outcome trajectories. The advances also consist of more powerful frames for understanding the ecology of disadvantage and the risk it poses for child outcomes. Most developmental researchers still tend to frame causal variables ultimately in terms of the dichotomy between social causation and social selection views, with a primary emphasis on the former. In part, this framing has reflected limitations of sample size and design, because the theoretical and empirical power of reciprocal selection models is clear (Kim et al., 2003). The conceptual advances that prompt such models include widespread acknowledgement of third variable problems in interpreting effects, of the clear need for multivariate approaches, and the need to pursue mechanisms and moderators of the relations between causal candidates and child outcomes. In the context of these advances, one of the core goals of our research program has been to construct robust representations of environmental adversity for disadvantaged families. Most of our research focuses on contextual co-factors at a family level (e.g., maternal

  7. Rice Bran Metabolome Contains Amino Acids, Vitamins & Cofactors, and Phytochemicals with Medicinal and Nutritional Properties.

    Science.gov (United States)

    Zarei, Iman; Brown, Dustin G; Nealon, Nora Jean; Ryan, Elizabeth P

    2017-12-01

    Rice bran is a functional food that has shown protection against major chronic diseases (e.g. obesity, diabetes, cardiovascular disease and cancer) in animals and humans, and these health effects have been associated with the presence of bioactive phytochemicals. Food metabolomics uses multiple chromatography and mass spectrometry platforms to detect and identify a diverse range of small molecules with high sensitivity and precision, and has not been completed for rice bran. This study utilized global, non-targeted metabolomics to identify small molecules in rice bran, and conducted a comprehensive search of peer-reviewed literature to determine bioactive compounds. Three U.S. rice varieties (Calrose, Dixiebelle, and Neptune), that have been used for human dietary intervention trials, were assessed herein for bioactive compounds that have disease control and prevention properties. The profiling of rice bran by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) identified 453 distinct phytochemicals, 209 of which were classified as amino acids, cofactors & vitamins, and secondary metabolites, and were further assessed for bioactivity. A scientific literature search revealed 65 compounds with health properties, 16 of which had not been previously identified in rice bran. This suite of amino acids, cofactors & vitamins, and secondary metabolites comprised 46% of the identified rice bran metabolome, which substantially enhanced our knowledge of health-promoting rice bran compounds provided during dietary supplementation. Rice bran metabolite profiling revealed a suite of biochemical molecules that can be further investigated and exploited for multiple nutritional therapies and medical food applications. These bioactive compounds may also be biomarkers of dietary rice bran intake. The medicinal compounds associated with rice bran can function as a network across metabolic pathways and this

  8. The primary haemostasis is more preserved in thrombocytopenic patients with liver cirrhosis than cancer

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Alnor, Anne B; Nybo, Mads

    2018-01-01

    aggregation using impedance aggregometry (Multiplate Analyzer), von Willebrand factor antigen (VWF:Ag), VWF:ristocetin-cofactor activity (VWF:RCo), activated partial thromboplastin time (aPTT), coagulation factor VIII, fibrinogen, and thrombin generation in adult hospitalized patients with platelet count less...... in patients with cirrhosis than cancer, 416 AU × min (IQR 257-676) versus 145 AU × min (IQR 50-326) for collagen-induced platelet aggregation, P time (aPTT), coagulation factor VIII, or thrombin generation between the patient groups...

  9. Origin of the Proton-transfer Step in the Cofactor-free (1H)-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

    Science.gov (United States)

    Hernandez-Ortega, Aitor; Quesne, Matthew G.; Bui, Soi; Heuts, Dominic P. H. M.; Steiner, Roberto A.; Heyes, Derren J.; de Visser, Sam P.; Scrutton, Nigel S.

    2014-01-01

    Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pKa value of ∼7.2 for WT HOD. A large solvent isotope effect was found, and the pKa value was shifted to ∼8.3 in D2O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation. PMID:24482238

  10. Cofactor of BRCA1: A new genetic marker for common malignant liver cancer

    Directory of Open Access Journals (Sweden)

    Editorial Office

    2016-08-01

    Full Text Available A new study has identified a vital gene in the pathogenesis and progression of liver cancer hepatocellular carcinoma (HCC, according to a team of biotechnology researchers at The American University in Cairo, Egypt, in a scientific paper published recently by AMOR. The study on human gene ‘Cofactor of BRCA1’ (dubbed COBRA1 and its potential role as a reliable cancer predictor for HCC is especially important due to the disease’s grim outlook. HCC is “ranked as the second most common cause of cancer-related deaths in the world in 2012,” the authors said. “Thus, it is considered as a highly aggressive cancer with poor prognosis,” they added. According to data from the Surveillance Epidemiology and End Results (SEER program, hepatocellular carcinoma accounts for 90% of all liver cancers worldwide. In the United States, HCC represents the fastest growing cause of cancer mortality overall and the second fastest growing cause of cancer deaths among women. Globally, the incidence of HCC in developing nations is over twice that of in developed countries – East Asia having highest incidence of HCC with the rate of 35 male cases per 100,000, followed by the continent of Africa. HCC mortality statistics in the developing countries is also more than double compared to the First World nations, with the annual loss of 33.5 and 23.73 lives per 100,000 in Asia and Africa, respectively. In addition, “HCC is usually diagnosed in the late stages of the tumor where, at some point, treatment is of limited efficacy. Thus, prognoses and follow-ups are necessary to regularly assess the patients and to predict any risks before the deterioration of patients’ condition,” said researcher Aya Youssef and her fellow team members. The behaviour of COBRA1 in the development and progression of several cancers has previously been studied and established, the researchers wrote. “For example, cell lines and tissues isolated from late-stage metastatic breast

  11. Protonation/reduction dynamics at the [4Fe-4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases.

    Science.gov (United States)

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Shulenina, Olga; Laun, Konstantin; Kertess, Leonie; Wittkamp, Florian; Apfel, Ulf-Peter; Happe, Thomas; Winkler, Martin; Haumann, Michael; Stripp, Sven T

    2018-01-31

    The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN - ) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN - vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN - infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

  12. Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

    Science.gov (United States)

    Xu, Jian-Zhong; Yang, Han-Kun; Liu, Li-Ming; Wang, Ying-Yu; Zhang, Wei-Guo

    2018-03-25

    l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L -1 ), carbon yield (from 0.35 to 0.44 g [g glucose] -1 ) and productivity (from 2.07 to 2.93 g L -1  hr -1 ) of l-lysine in JL-6 ΔdapB::Ec-dapB C115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB C115G,G116C , followed by JL-6 Cg-dapB C37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapB C46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity. © 2018 Wiley Periodicals, Inc.

  13. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

    Science.gov (United States)

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-02-27

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

    Science.gov (United States)

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-01

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

  15. An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Singh, Shalini; Mörgelin, Matthias; Schmidtchen, Artur; Malmsten, Martin

    2014-05-01

    Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. AMOEBA Polarizable Force Field Parameters of the Heme Cofactor in Its Ferrous and Ferric Forms.

    Science.gov (United States)

    Wu, Xiaojing; Clavaguera, Carine; Lagardère, Louis; Piquemal, Jean-Philip; de la Lande, Aurélien

    2018-04-16

    We report the first parameters of the heme redox cofactors for the polarizable AMOEBA force field in both the ferric and ferrous forms. We consider two types of complexes, one with two histidine side chains as axial ligands and one with a histidine and a methionine side chain as ligands. We have derived permanent multipoles from second-order Møller-Plesset perturbation theory (MP2). The sets of parameters have been validated in a first step by comparison of AMOEBA interaction energies of heme and a collection of biologically relevant molecules with MP2 and Density Functional Theory (DFT) calculations. In a second validation step, we consider interaction energies with large aggregates comprising around 80 H 2 O molecules. These calculations are repeated for 30 structures extracted from semiempirical PM7 DM simulations. Very encouraging agreement is found between DFT and the AMOEBA force field, which results from an accurate treatment of electrostatic interactions. We finally report long (10 ns) MD simulations of cytochromes in two redox states with AMOEBA testing both the 2003 and 2014 AMOEBA water models. These simulations have been carried out with the TINKER-HP (High Performance) program. In conclusion, owing to their ubiquity in biology, we think the present work opens a wide array of applications of the polarizable AMOEBA force field on hemeproteins.

  17. Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

    Science.gov (United States)

    Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

    2018-05-15

    Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

  18. Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites

    Science.gov (United States)

    Mortusewicz, Oliver; Roth, Wera; Li, Na; Cardoso, M. Cristina; Meisterernst, Michael; Leonhardt, Heinrich

    2008-01-01

    The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and γH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair. PMID:19047459

  19. The geochemical record of the ancient nitrogen cycle, nitrogen isotopes, and metal cofactors.

    Science.gov (United States)

    Godfrey, Linda V; Glass, Jennifer B

    2011-01-01

    The nitrogen (N) cycle is the only global biogeochemical cycle that is driven by biological functions involving the interaction of many microorganisms. The N cycle has evolved over geological time and its interaction with the oxygen cycle has had profound effects on the evolution and timing of Earth's atmosphere oxygenation (Falkowski and Godfrey, 2008). Almost every enzyme that microorganisms use to manipulate N contains redox-sensitive metals. Bioavailability of these metals has changed through time as a function of varying redox conditions, and likely influenced the biological underpinnings of the N cycle. It is possible to construct a record through geological time using N isotopes and metal concentrations in sediments to determine when the different stages of the N cycle evolved and the role metal availability played in the development of key enzymes. The same techniques are applicable to understanding the operation and changes in the N cycle through geological time. However, N and many of the redox-sensitive metals in some of their oxidation states are mobile and the isotopic composition or distribution can be altered by subsequent processes leading to erroneous conclusions. This chapter reviews the enzymology and metal cofactors of the N cycle and describes proper utilization of methods used to reconstruct evolution of the N cycle through time. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production.

    Science.gov (United States)

    Zhang, Liang; Tang, Yan; Guo, Zhongpeng; Shi, Guiyang

    2013-10-01

    Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.

  2. DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

    Science.gov (United States)

    Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

    2016-11-02

    Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Biocatalytic hydroxylation of n-butane with in situ cofactor regeneration at low temperature and under normal pressure

    Directory of Open Access Journals (Sweden)

    Svenja Staudt

    2012-02-01

    Full Text Available The hydroxylation of n-alkanes, which proceeds in the presence of a P450-monooxygenase advantageously at temperatures significantly below room temperature, is described. In addition, an enzymatic hydroxylation of the “liquid gas” n-butane with in situ cofactor regeneration, which does not require high-pressure conditions, was developed. The resulting 2-butanol was obtained as the only regioisomer, at a product concentration of 0.16 g/L.

  4. Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances.

    Directory of Open Access Journals (Sweden)

    Du Toit W P Schabort

    Full Text Available The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production.

  5. Absorption and emission spectroscopic characterization of BLUF protein Slr1694 from Synechocystis sp. PCC6803 with roseoflavin cofactor.

    Science.gov (United States)

    Zirak, P; Penzkofer, A; Mathes, T; Hegemann, P

    2009-11-09

    The wild-type BLUF protein Slr1694 from Synechocystis sp. PCC6803 (BLUF=blue-light sensor using FAD) has flavin adenosine dinucleotide (FAD) as natural cofactor. This light sensor causes positive phototaxis of the marine cyanobacterium. In this study the FAD cofactor of the wild-type Slr1694 was replaced by roseoflavin (RoF) and the roseoflavin derivatives RoFMN and RoFAD during heterologous expression in a riboflavin auxotrophic E. coli strain. An absorption and emission spectroscopic characterization of the cofactor-exchanged-Slr1694 (RoSlr) was carried out both under dark conditions and under illuminated conditions. The behaviour of RoF embedded in RoSlr in aqueous solution at pH 8 is compared with the behaviour of RoF in aqueous solution. The fluorescence of RoF and RoSlr is quenched by photo-induced twisted intra-molecular charge transfer at room temperature with stronger effect for RoF. The fluorescence quenching is diminished at liquid nitrogen temperature. Light exposure of RoSlr causes irreversible conversion of the protein embedded roseoflavins to 8-methylamino-flavins, 8-dimethylamino-lumichrome and 8-methylamino-lumichrome.

  6. Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

    Science.gov (United States)

    Chocklett, Samuel W; Sobrado, Pablo

    2010-08-10

    Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

  7. Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

    Science.gov (United States)

    Ren, Weitong; Ji, Dongqing; Xu, Xiulian

    2018-01-01

    The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

  8. Cofactors involved in light-driven charge separation in photosystem I identified by subpicosecond infrared spectroscopy.

    Science.gov (United States)

    Di Donato, Mariangela; Stahl, Andreas D; van Stokkum, Ivo H M; van Grondelle, Rienk; Groot, Marie-Louise

    2011-02-01

    Photosystem I is one of the key players in the conversion of solar energy into chemical energy. While the chlorophyll dimer P(700) has long been identified as the primary electron donor, the components involved in the primary charge separation process in PSI remain undetermined. Here, we have studied the charge separation dynamics in Photosystem I trimers from Synechococcus elongatus by femtosecond vis-pump/mid-infrared-probe spectroscopy upon excitation at 700, 710, and 715 nm. Because of the high specificity of the infrared region for the redox state and small differences in the molecular structure of pigments, we were able to clearly identify specific marker bands indicating chlorophyll (Chl) oxidation. Magnitudes of chlorophyll cation signals are observed to increase faster than the time resolution of the experiment (~0.2 ps) upon both excitation conditions: 700 nm and selective red excitation. Two models, involving either ultrafast charge separation or charge transfer character of the red pigments in PSI, are discussed to explain this observation. A further increase in the magnitudes of cation signals on a subpicosecond time scale (0.8-1 ps) indicates the formation of the primary radical pair. Evolution in the cation region with time constants of 7 and 40 ps reveals the formation of the secondary radical pair, involving a secondary electron donor. Modeling of the data allows us to extract the spectra of the two radical pairs, which have IR signatures consistent with A+A₀- and P₇₀₀+A₁-. We conclude that the cofactor chlorophyll A acts as the primary donor in PSI. The existence of an equilibrium between the two radical pairs we interpret as concerted hole/electron transfer between the pairs of electron donors and acceptors, until after 40 ps, relaxation leads to a full population of the P₇₀₀+A₁. radical pair.

  9. Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

    Directory of Open Access Journals (Sweden)

    Weitong Ren

    Full Text Available The HIV-1 nucleocapsid 7 (NCp7 plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3 recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

  10. Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

    Science.gov (United States)

    Levillain, Florence; Poquet, Yannick; Mallet, Ludovic; Mazères, Serge; Marceau, Michael; Brosch, Roland; Bange, Franz-Christoph; Supply, Philip; Magalon, Axel; Neyrolles, Olivier

    2017-11-01

    The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

  11. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

    Science.gov (United States)

    Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

    2016-02-22

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

  12. Tubulin binding cofactor C (TBCC) suppresses tumor growth and enhances chemosensitivity in human breast cancer cells

    International Nuclear Information System (INIS)

    Hage-Sleiman, Rouba; Herveau, Stéphanie; Matera, Eva-Laure; Laurier, Jean-Fabien; Dumontet, Charles

    2010-01-01

    Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers. We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells. TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts. These

  13. The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

    Science.gov (United States)

    Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

    2004-05-01

    Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

  14. The mechanism of Torsin ATPase activation.

    Science.gov (United States)

    Brown, Rebecca S H; Zhao, Chenguang; Chase, Anna R; Wang, Jimin; Schlieker, Christian

    2014-11-11

    Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

  15. Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

    Science.gov (United States)

    Augustin, Peter; Toplak, Marina; Fuchs, Katharina; Gerstmann, Eva Christine; Prassl, Ruth; Winkler, Andreas; Macheroux, Peter

    2018-01-01

    The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria. PMID:29301933

  16. Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

    Science.gov (United States)

    Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

    2016-10-25

    Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

  17. Niveles plasmáticos e interacciones del sistema cofactor 2 de la heparina-trombina-dermatan sulfato

    OpenAIRE

    Rossi, Eleonora Beatriz

    1999-01-01

    El Cofactor II de la Heparina (HCII) es un inhibidor fisiológico del sistema de coagulación, miembro de la familia de serpinas. Inhibe específicamente trombina, una enzima clave del sistema hemostático. La capacidad del HCII de inhibir trombina es potenciada mas de 1000 veces por la presencia de glicosaminoglicano el Dermatán Sulfato (DS),. Aún no está claramente definido el papel que desempeña el HCII en la fisiología de la Hemostasia, postulándose su deficiencia corno leve factor de riesgo ...

  18. Integrating biocompatible chemistry and manipulating cofactor partitioning in metabolically engineeredLactococcus lactisfor fermentative production of (3S)-acetoin

    DEFF Research Database (Denmark)

    Liu, Jianming; Solem, Christian; Jensen, Peter Ruhdal

    2016-01-01

    Biocompatible chemistry (BC), i.e. non-enzymatic chemical reactions compatible with living organisms, is increasingly used in conjunction with metabolically engineered microorganisms for producing compounds that do not usually occur naturally. Here we report production of one such compound, (3S......)-acetoin, a valuable precursor for chiral synthesis, using a metabolically engineered Lactococcus lactis strain growing under respiratory conditions with ferric iron serving as a BC component. The strain used has all competing product pathways inactivated, and an appropriate cofactor balance is achieved by fine...

  19. Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

    International Nuclear Information System (INIS)

    Azim, N.; Deery, E.; Warren, M. J.; Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B.; Coker, A.; Wood, S. P.; Akhtar, M.

    2014-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form

  20. The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Keith S Wong

    Full Text Available MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37 °C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I. Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.

  1. Cofactor Balance by Nicotinamide Nucleotide Transhydrogenase (NNT) Coordinates Reductive Carboxylation and Glucose Catabolism in the Tricarboxylic Acid (TCA) Cycle*♦

    Science.gov (United States)

    Gameiro, Paulo A.; Laviolette, Laura A.; Kelleher, Joanne K.; Iliopoulos, Othon; Stephanopoulos, Gregory

    2013-01-01

    Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)+ cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)+ ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle. PMID:23504317

  2. Homeotic function of Drosophila Bithorax-Complex miRNAs mediates fertility by restricting multiple Hox genes and TALE cofactors in the central nervous system

    Science.gov (United States)

    Garaulet, Daniel L.; Castellanos, Monica; Bejarano, Fernando; Sanfilippo, Piero; Tyler, David M.; Allan, Douglas W.; Sánchez-Herrero, Ernesto; Lai, Eric C.

    2014-01-01

    The Drosophila Bithorax-Complex (BX-C) Hox cluster contains a bidirectionally-transcribed miRNA locus, and a deletion mutant (∆mir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the central nervous system. ∆mir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, since sterility of ∆mir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in ∆mir females, and substantially rescued by heterozygosity of ∆mir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation, and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior. PMID:24909902

  3. Conformational control of cofactors in nature: The effect of methoxy group orientation on the electronic structure of ubisemiquinone

    Science.gov (United States)

    De Almeida, Wagner B.; O'Malley, Patrick J.

    2018-03-01

    Ubiquinone is the key electron and proton transfer agent in biology. Its mechanism involves the formation of its intermediate one-electron reduced form, the ubisemiquinone radical. This is formed in a protein-bound form which permits the semiquinone to vary its electronic and redox properties. This can be achieved by hydrogen bonding acceptance by one or both oxygen atoms or as we now propose by restricted orientations for the methoxy groups of the headgroup. We show how the orientation of the two methoxy groups of the quinone headgroup affects the electronic structure of the semiquinone form and demonstrate a large dependence of the ubisemiquinone spin density distribution on the orientation each methoxy group takes with respect to the headgroup ring plane. This is shown to significantly modify associated hyperfine couplings which in turn needs to be accounted for in interpreting experimental values in vivo. The study uncovers the key potential role the methoxy group orientation can play in controlling the electronic structure and spin density of ubisemiquinone and provides an electronic-level insight into the variation in electron affinity and redox potential of ubiquinone as a function of the methoxy orientation. Taken together with the already known influence of cofactor conformation on heme and chlorophyll electronic structure, it reveals a more widespread role for cofactor conformational control of electronic structure and associated electron transfer in biology.

  4. Cofactor and CO2 donor regulation involved in reductive routes for polymalic acid production by Aureobasidium pullulans CCTCC M2012223.

    Science.gov (United States)

    Zou, Xiang; Tu, Guangwei; Zan, Zhanquan

    2014-10-01

    Polymalic acid (PMA) is a water-soluble polyester with many attractive properties for biomedical application. Its monomer L-malic acid is widely used in the food industry and also a potential C4 platform chemical. Cofactor and CO2 donor involved in the reductive routes were investigated for PMA production by Aureobasidium pullulans. Biotin as the key cofactor of pyruvate carboxylase was favor for the PMA biosynthesis. Na2CO3 as CO2 donor can obviously improved PMA titer when compared with no CO2 supplier NaOH, and also exhibit more advantages than the other donor CaCO3 because of its water-soluble characteristic. A combinational process with addition of biotin 70 mg/L and Na2CO3 as the CO2 donor was scaled-up in 50 L fermentor, achieving the high product 34.3 g/L of PMA and productivity of 0.41 g/L h. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application.

  5. Purification, crystallization and preliminary crystallographic analysis of a multiple cofactor-dependent DNA ligase from Sulfophobococcus zilligii

    International Nuclear Information System (INIS)

    Supangat, Supangat; An, Young Jun; Sun, Younguk; Kwon, Suk-Tae; Cha, Sun-Shin

    2010-01-01

    A recombinant multiple cofactor-dependent DNA ligase from S. zilligii has been purified and crystallized. X-ray diffraction data were collected to 2.9 Å resolution and the crystals belonged to space group P1. A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml −1 ) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 63.7, b = 77.1, c = 77.8 Å, α = 83.4, β = 82.4, γ = 74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%

  6. Duplicated Gephyrin Genes Showing Distinct Tissue Distribution and Alternative Splicing Patterns Mediate Molybdenum Cofactor Biosynthesis, Glycine Receptor Clustering, and Escape Behavior in Zebrafish*

    Science.gov (United States)

    Ogino, Kazutoyo; Ramsden, Sarah L.; Keib, Natalie; Schwarz, Günter; Harvey, Robert J.; Hirata, Hiromi

    2011-01-01

    Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABAA receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish. PMID:20843816

  7. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

    Energy Technology Data Exchange (ETDEWEB)

    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N. (Toronto)

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  8. Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1[alpha

    Energy Technology Data Exchange (ETDEWEB)

    Rho, H.; Jones, C.N.; Rose, R.B. (NCSU)

    2010-12-07

    The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1{alpha} (hepatocyte nuclear factor-1{alpha}), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1{alpha} complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1{alpha} through simple mixing. To further investigate regulation of DCoH/HNF-1{alpha} complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1{alpha}. The 1.8 {angstrom} resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1{alpha} complex formation are discussed.

  9. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf

    2010-10-21

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  10. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  11. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

    2010-01-01

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  12. Relative contributions of decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 in the protection of melanocytes from homologous complement

    NARCIS (Netherlands)

    Venneker, G. T.; Vodegel, R. M.; Okada, N.; Westerhof, W.; Bos, J. D.; Asghar, S. S.

    1998-01-01

    Complement regulatory molecules, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, protect body cells from autologous complement. They have wide tissue distribution but nothing is known about the expression of these molecules on human melanocytes. Since melanocytes are lysed

  13. S-Adenosyl-S-carboxymethyl-l-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA

    International Nuclear Information System (INIS)

    Byrne, Robert T.; Whelan, Fiona; Aller, Pierre; Bird, Louise E.; Dowle, Adam; Lobley, Carina M. C.; Reddivari, Yamini; Nettleship, Joanne E.; Owens, Raymond J.; Antson, Alfred A.; Waterman, David G.

    2013-01-01

    The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group. Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo 5 U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo 5 U and was annotated as an S-adenosyl-l-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-l-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry http://scripts.iucr.org/cgi-bin/cr.cgi?rm, suggests that the active site contains SCM-SAH and not SAM

  14. S-Adenosyl-S-carboxymethyl-l-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA

    Energy Technology Data Exchange (ETDEWEB)

    Byrne, Robert T.; Whelan, Fiona [University of York, Heslington YO10 5DD (United Kingdom); Aller, Pierre [Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE (United Kingdom); Bird, Louise E. [OPPF-UK, Research Complex at Harwell, R92 Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0FA (United Kingdom); Oxford University, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Dowle, Adam [University of York, Heslington YO10 5DD (United Kingdom); Lobley, Carina M. C. [Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE (United Kingdom); Reddivari, Yamini; Nettleship, Joanne E.; Owens, Raymond J. [OPPF-UK, Research Complex at Harwell, R92 Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0FA (United Kingdom); Oxford University, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Antson, Alfred A. [University of York, Heslington YO10 5DD (United Kingdom); Waterman, David G., E-mail: david.waterman@stfc.ac.uk [STFC, Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0FA (United Kingdom); University of York, Heslington YO10 5DD (United Kingdom)

    2013-06-01

    The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group. Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo{sup 5}U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo{sup 5}U and was annotated as an S-adenosyl-l-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-l-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry http://scripts.iucr.org/cgi-bin/cr.cgi?rm, suggests that the active site contains SCM-SAH and not SAM.

  15. Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

    Science.gov (United States)

    Revsbech, Inge G; Malte, Hans; Fröbert, Ole; Evans, Alina; Blanc, Stéphane; Josefsson, Johan; Fago, Angela

    2013-01-01

    During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

  16. Ubxd1 is a novel co-factor of the human p97 ATPase

    DEFF Research Database (Denmark)

    Madsen, Louise; Andersen, Katrine M; Prag, Søren

    2008-01-01

    The AAA ATPase complex known as p97 or VCP in mammals and Cdc48 in yeast is connected to a multitude of cellular pathways, including membrane fusion, protein folding, protein degradation and activation of membrane-bound transcription factors. The mechanism by which p97 participates in such a broad...

  17. X-ray absorption spectroscopy on the calcium cofactor to the manganese cluster in photosynthetic oxygen evolution

    Energy Technology Data Exchange (ETDEWEB)

    Cinco, Roehl M. [Univ. of California, Berkeley, CA (United States)

    1999-12-01

    Along with Mn, calcium and chloride ions are necessary cofactors for oxygen evolution in Photosystem II (PS II). To further test and verify whether Ca is close to the Mn cluster, the authors substituted strontium for Ca and probed from the Sr point of view for any nearby Mn. The extended X-ray absorption fine structure (EXAFS) of Sr-reactivated PS II indicates major differences between the intact and NH2OH-treated samples. In intact samples, the Fourier transform of the Sr EXAFS shows a Fourier peak that is missing in inactive samples. This peak II is best simulated by two Mn neighbors at a distance of 3.5 Angstrom, confirming the proximity of Ca (Sr) cofactor to the Mn cluster. In addition, polarized Sr EXAFS on oriented Sr-reactivated samples shows this peak II is dichroic: large magnitude at 10 degrees (angle between the PS II membrane normal and the x-ray electric field vector) and small at 80 degrees. Analysis of the dichroism yields the relative angle between the Sr-Mn vector and membrane normal (23 degrees ± 4 degrees), and the isotropic coordination number for these layered samples. X-ray absorption spectroscopy has also been employed to assess the degree of similarity between the manganese cluster in PS II and a family of synthetic manganese complexes containing the distorted cubane [Mn4O3X] core (X = benzoate, acetate, methoxide, hydroxide, azide, fluoride, chloride or bromide). In addition, Mn4O3Cl complexes containing three or six terminal Cl ligands at three of the Mn were included in this study. The EXAFS method detects the small changes in the core structures as X is varied in this series, and serves to exclude these distorted cubanes of C3v symmetry as a topological model for the Mn catalytic cluster. The sulfur K-edge x-ray absorption near-edge structure (XANES) spectra for the amino acids cysteine, methionine, their corresponding oxidized forms cystine and methionine sulfoxide, and

  18. Activation of human factor V by factor Xa and thrombin

    International Nuclear Information System (INIS)

    Monkovic, D.D.; Tracy, P.B.

    1990-01-01

    The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of 125 I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of M r 220,000 and 105,000. Although thrombin cleaved the M r 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the M r 220,000 peptide. The factor Xa dependent functional assessment of 125 I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the M r 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin

  19. Identification of cofactor and herbicide binding domains in acetolactate synthase by bromopyruvate modification

    International Nuclear Information System (INIS)

    Van Dyk, D.E.; Schloss, J.V.

    1987-01-01

    Bromopyruvate is an affinity label for acetolactate synthase isozyme II from Salmonella typhimurium (ALSII). The concentration of bromopyruvate giving half-maximal inactivation is 0.1 mM, and the maximal rate of inactivation is 0.56 hr -1 . Inactivation with [ 14 C]bromopyruvate is associated with the incorporation of 4 molecules of reagent per active site lost. Two cysteinyl residues are modified extremely rapidly, with no loss of enzymatic activity, as judged by quenching the reaction with thiol after its initial phase. Inactivation is a consequence of the additional two moles of reagent incorporated per mole of protomer. The additional incorporation is divided between one major and two minor sites of modification. Substantial protection against inactivation is afforded by FAD, with virtually complete protection provided by a mixture of FAD and thiamine pyrophosphate (TPP). The major site of modification, protected by FAD, is cysteinyl residue number67, based upon amino acid sequence analysis of the purified tryptic peptide that encompasses this site. The remaining site of modification, protected by TPP, is associated with cysteinyl residue number44. Both sites of modification are afforded protection by the sulfonylurea herbicide sulfometuron methyl (SM). Although inactivation by bromopyruvate exhibits rate saturation, indicating binding as a prerequisite to inactivation, neither pyruvate nor α-ketobutyrate prevent modification of the enzyme by bromopyruvate. Thus, it would appear that the bromopyruvate binding site is not the site normally occupied by substrate

  20. Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean.

    Science.gov (United States)

    Almonte, Maribel; Albero, Ginesa; Molano, Mónica; Carcamo, César; García, Patricia J; Pérez, Gonzalo

    2008-08-19

    The incidence of cervical cancer in Latin America and the Caribbean (LAC) is among the highest in the world. Because there are major demographic shifts happening in LAC countries (population growth, urbanization and ageing) cervical cancer incidence and mortality will likely continue to be a significant public health problem. Overall human papillomavirus (HPV) prevalence in the LAC general population has been found to be 2-fold higher than the average worldwide prevalence. The large HPV and cancer burden may be explained by the highly prevalent HPV variants of HPV types -16 and 18, which have an increased oncogenic potential. Given the major mode of transmission of genital HPV is sexual, certain, patterns of sexual behaviour (early age at first sexual intercourse, number of sexual partners and sexual behaviour of the partner) are associated with an increased risk of HPV genital acquisition. Although HPV infection is necessary for carcinogenesis, certain co-factors (high parity, long term use of oral contraceptives, smoking and co-infection with the human immunodeficiency virus (HIV)) help in the progression from infection to cancer. Many studies that have contributed to this evidence have been carried out in LAC and are reviewed and summarised in this article. Since HPV vaccines will likely take years to implement, and many more years to show impact on disease, cervical cancer screening programmes remain as the key intervention to control disease in LAC in the years to come.

  1. Development of CHARMM-Compatible Force-Field Parameters for Cobalamin and Related Cofactors from Quantum Mechanical Calculations.

    Science.gov (United States)

    Pavlova, Anna; Parks, Jerry M; Gumbart, James C

    2018-02-13

    Corrinoid cofactors such as cobalamin are used by many enzymes and are essential for most living organisms. Therefore, there is broad interest in investigating cobalamin-protein interactions with molecular dynamics simulations. Previously developed parameters for cobalamins are based mainly on crystal structure data. Here, we report CHARMM-compatible force field parameters for several corrinoids developed from quantum mechanical calculations. We provide parameters for corrinoids in three oxidation states, Co 3+ , Co 2+ , and Co 1+ , and with various axial ligands. Lennard-Jones parameters for the cobalt center in the Co(II) and Co(I) states were optimized using a helium atom probe, and partial atomic charges were obtained with a combination of natural population analysis (NPA) and restrained electrostatic potential (RESP) fitting approaches. The Force Field Toolkit was used to optimize all bonded terms. The resulting parameters, determined solely from calculations of cobalamin alone or in water, were then validated by assessing their agreement with density functional theory geometries and by analyzing molecular dynamics simulation trajectories of several corrinoid proteins for which X-ray crystal structures are available. In each case, we obtained excellent agreement with the reference data. In comparison to previous CHARMM-compatible parameters for cobalamin, we observe a better agreement for the fold angle and lower RMSD in the cobalamin binding site. The approach described here is readily adaptable for developing CHARMM-compatible force-field parameters for other corrinoids or large biomolecules.

  2. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.; Ludwig, Martha L.; Matthews, Rowena G. (Michigan)

    2008-07-08

    B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.

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

    Directory of Open Access Journals (Sweden)

    Silke Leimkühler

    2017-01-01

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

  4. The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1

    International Nuclear Information System (INIS)

    Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Heng Koh, Choon; Zimmermann, Holger; Maolin Yang, Christopher; Guan Sim, Khe; I-Hong Hsu, Stephen; Bernard, Hans-Ulrich

    2003-01-01

    The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ('low-risk types'), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ('high-risk types'), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

  5. Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.

    Directory of Open Access Journals (Sweden)

    Maria Zhadina

    2010-10-01

    Full Text Available The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L- domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1. It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV, where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

  6. The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

    Energy Technology Data Exchange (ETDEWEB)

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dow Street, Dundee DD1 5EH, Scotland (United Kingdom)

    2015-04-21

    The structure of a tubulin-binding cofactor from L. major is reported and compared with yeast, plant and human orthologues. Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area.

  7. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

  8. Differential developmental expression of transcription factors GATA-4 and GATA-6, their cofactor FOG-2 and downstream target genes in testicular carcinoma in situ and germ cell tumors

    DEFF Research Database (Denmark)

    Salonen, Jonna; Rajpert-De Meyts, E; Mannisto, Susanna

    2010-01-01

    Testicular germ cell cancer is the most common malignancy among young males. The pre-invasive precursor, carcinoma in situ testis (CIS), presumably originates from arrested and transformed fetal gonocytes. Given that GATA transcription factors have essential roles in embryonic and testicular deve...... development, we explored the expression of GATA-4, GATA-6, cofactor friend of GATA (FOG)-2, and downstream target genes during human testis development and addressed the question whether changes in this pathway may contribute to germ cell neoplasms....

  9. Expression, purification, crystallization and preliminary phasing of the heteromerization domain of the tRNA-export and aminoacylation cofactor Arc1p from yeast

    International Nuclear Information System (INIS)

    Simader, Hannes; Suck, Dietrich

    2006-01-01

    The heteromerization domain of an aminoacyl-tRNA synthetase cofactor from yeast was crystallized, complete selenomethionine MAD data were collected to 2.8 Å resolution and preliminary phasing reveals the presence of 20 monomers in the asymmetric unit. Eukaryotic aminoacyl-tRNA synthetases (aaRSs) must be integrated into an efficient tRNA-export and shuttling machinery. This is reflected by the presence of additional protein–protein interaction domains and a correspondingly higher degree of complex formation in eukaryotic aaRSs. However, the structural basis of interaction between eukaryotic aaRSs and associated protein cofactors has remained elusive. The N-terminal heteromerization domain of the tRNA aminoacylation and export cofactor Arc1p has been cloned from yeast, expressed and purified. Crystals have been obtained belonging to space group C2, with unit-cell parameters a = 222.32, b = 89.46, c = 126.79 Å, β = 99.39°. Calculated Matthews coefficients are compatible with the presence of 10–25 monomers in the asymmetric unit. A complete multiple-wavelength anomalous dispersion data set has been collected from a selenomethionine-substituted crystal at 2.8 Å resolution. Preliminary phasing reveals the presence of 20 monomers organized in five tetramers per asymmetric unit

  10. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    Directory of Open Access Journals (Sweden)

    Michael Katzberg

    2010-04-01

    Full Text Available The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S-hexanediol and the γ-hydroxyketone (5S-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%. This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

  11. Functional analysis of the interaction of the human immunodeficiency virus type 1 Rev nuclear export signal with its cofactors

    International Nuclear Information System (INIS)

    Kiss, A.; Li, L.; Gettemeier, T.; Venkatesh, L.K.

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export of viral RNAs involves the interaction of its leucine-rich nuclear export sequence (NES) with nuclear cofactors. In yeast two-hybrid screens of a human lymph node derived cDNA expression library, we identified the human nucleoporin Nup98 as a highly specific and potent interactor of the Rev NES. Using an extensive panel of nuclear export positive and negative mutants of the functionally homologous NESs of the HIV-1 Rev, human T cell leukemia virus type 1 (HTLV-1) Rex, and equine infectious anemia virus (EIAV) Rev proteins, physiologically significant interaction of hNup98 with the various NESs was demonstrated. Missense mutations in the yeast nuclear export factor Crm1p that abrogated Rev NES interaction with the XXFG repeat-containing nucleoporin, Rab/hRIP, had minimal effects on the interaction of GLFG repeat-containing hNup98. Functional analysis of Nup98 domains required for nuclear localization demonstrated that the entire ORF was required for efficient incorporation into the nuclear envelope. A putative nuclear localization signal was identified downstream of the GLFG repeat region. Whereas overexpression of both full-length Nup98 and the amino-terminal GLFG repeat region, but not the unique carboxy-terminal region, induced significant suppression of HIV unspliced RNA export, lower levels of exogenous Nup98 expression resulted in a relatively modest increase in unspliced RNA export. These results suggest a physiological role for hNup98 in modulating Rev-dependent RNA export during HIV infection

  12. Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population.

    Science.gov (United States)

    Wang, Zongkui; Dou, Miaomiao; Du, Xi; Ma, Li; Sun, Pan; Cao, Haijun; Ye, Shengliang; Jiang, Peng; Liu, Fengjuan; Lin, Fangzhao; Zhang, Rong; Li, Changqing

    2017-01-01

    ABO blood group is a hereditary factor of plasma levels of coagulation factor VIII (FVIII) and von Willebrand factor (VWF). Age and gender have been shown to influence FVIII, VWF, fibrinogen (Fbg), and ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 motif, 13). We investigated the effects of ABO type, age, and gender on plasma levels of FVIII, Fbg, VWF, and ADAMTS13 in a Chinese population. A total of 290 healthy volunteers were eligible for this study. ABO blood group was determined by indirect technique. FVIII:C and Fbg were measured by clotting assays. VWF antigen (VWF:Ag), collagen-binding activity (VWF:CBA), and ADAMTS13 antigen were assessed by ELISA, whereas VWF ristocetin cofactor activity (VWF:Rcof) was performed by agglutination of platelets with ristocetin. Mean FVIII:C and VWF levels (VWF:Ag, VWF:CBA, and VWF:Rcof) were significantly higher in non-O than in O type subjects ( p  blood group, age, and gender showed different effects on plasma levels of FVIII:C, Fbg, VWF:Ag, VWF:CBA, VWF:Rcof, and ADAMTS13 antigen. These new data on a Chinese population are quite helpful to compare with other ethnic groups.

  13. Diagnosis and management of von Willebrand disease: a developing country perspective.

    Science.gov (United States)

    Nair, Sukesh Chandran; Viswabandya, Auro; Srivastava, Alok

    2011-07-01

    Special challenges exist in the management of patients with von Willebrand disease (VWD) because of limitations in diagnostic facilities and therapeutic options. However, even within these limitations, it is possible to establish comprehensive services for this condition. Our data show that among 202 patients with VWD, 107 were type 3, 62 were type 1, and the others different categories of type 2. Basic tests such as bleeding time and activated partial thromboplastin time with factor (F)VIII coagulant are able to diagnose most of those with severe disease. We have been able to adapt the specific tests such as von Willebrand factor (VWF) ristocetin cofactor and VWF antigen from the tedious batched manual methods to cost-effective automated methods on advanced coagulometers. Discriminatory tests such as VWF collagen binding, VWF:FVIIIB, ristocetin-induced platelet agglutination (RIPA) are done in batches. Therapeutic options and for the treatment of bleeding include desmopressin, cryoprecipitate, and intermediate purity VWF-containing clotting factor concentrates. Tranexamic acid is also widely used as well as hormonal therapy for menorrhagia. We have also shown that modest doses of intermediate purity FVIII (Koate DVI; Talecris Biotherapeutics, Raleigh, NC, USA) at 35 IU/kg preoperatively and 10 to 20 IU/kg after that are sufficient for surgical hemostasis in these patients. © Thieme Medical Publishers.

  14. A study of the effect of ethamsylate (Dicynene) on the bleeding time, von Willebrand factor level and fibrinolysis in patients with von Willebrand's disease.

    Science.gov (United States)

    Hutton, R A; Hales, M; Kernoff, P B

    1988-12-22

    Nine patients with clinically moderate or severe Type I von Willebrand's disease were treated for 2 weeks with ethamsylate (2 g/day in four equal doses) and with a matched placebo in a randomised double-blind trial. Template bleeding time, von Willebrand factor activity (ristocetin co-factor) and antigen, euglobulin lysis time and type I tissue plasminogen activator inhibitor were determined before and at the end of each treatment period. None of these parameters showed any significant change attributable to ethamsylate. Thus, despite the fact that five patients thought subjectively that their bleeding symptoms improved during ethamsylate treatment compared to only one while on placebo, we obtained no evidence that the drug was of benefit to patients with von Willebrand's disease.

  15. Dynamic mechanistic modeling of the multienzymatic one-pot reduction of dehydrocholic acid to 12-keto ursodeoxycholic acid with competing substrates and cofactors.

    Science.gov (United States)

    Sun, Boqiao; Hartl, Florian; Castiglione, Kathrin; Weuster-Botz, Dirk

    2015-01-01

    Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers.

  16. Pharmacoepidemiological assessment of adherence and influencing co-factors among primary open-angle glaucoma patients-An observational cohort study.

    Directory of Open Access Journals (Sweden)

    Stefanie Frech

    Full Text Available The goal of this study was to assess the adherence of primary open-angle glaucoma (POAG patients to medication, and to determine co-factors influencing adherence, using a representative sample of members of the largest German public health insurer. The observational cohort study was based on a longitudinal data set from 2010-2013 and included 250,000 insured persons aged 50 and older with 10,120 diagnosed POAG patients. Uni- and multivariate analysis was performed to investigate several aspects of glaucoma, such as prevalence, adherence, and co-factors influencing adherence. The main outcome measured adherence with prescriptions filled within a year. Multivariate panel regression analysis was used to determine the co-factors influencing this adherence. Prevalence of POAG was 3.36% [CI: 3.28-3.43%], with 2.91% [CI: 2.81-3.01%] for males and 3.71% [CI: 3.61-3.81%] for females, increasing with age. The mean level of adherence in terms of prescriptions filled was 66.5% [CI: 65.50-67.60%]. The results of this analysis revealed a significant influence of age, duration of the disease, care need, distance to death, and multimorbidity as co-factors of non-adherence, whereas gender had no influence. The analysis provided detailed information about POAG health care aspects concerning prevalence and adherence. The most endangered risk groups for non-adherence were patients aged 50-59, patients older than 80 years, patients with a longer duration of POAG, patients with care needs, and patients with three or more severe diseases in addition to glaucoma. To know the predictors responsible for an increased risk to develop POAG is of importance for all persons involved in health care management. Therefore effective strategies to increase awareness of patients and medical care personnel about non-adherence and the importance of a regular and continuous medication to avoid further nerve fiber damage and possible blindness have to be developed.

  17. Synthesis and characterization of sulfur-voided cubanes. Structural analogues for the MoFe(3)S(3) subunit in the nitrogenase cofactor.

    Science.gov (United States)

    Coucouvanis, Dimitri; Han, Jaehong; Moon, Namdoo

    2002-01-16

    A new class of Mo/Fe/S clusters with the MoFe(3)S(3) core has been synthesized in attempts to model the FeMo-cofactor in nitrogenase. These clusters are obtained in reactions of the (Cl(4)-cat)(2)Mo(2)Fe(6)S(8)(PR(3))(6) [R = Et (I), (n)Pr (II)] clusters with CO. The new clusters include those preliminarily reported: (Cl(4)-cat)MoFe(3)S(3)(PEt(3))(2)(CO)(6) (III), (Cl(4)-cat)(O)MoFe(3)S(3)(PEt(3))(3)(CO)(5) (IV), (Cl(4)-cat)(Pyr)MoFe(3)S(3)(PEt(3))(2)(CO)(6) (VI), and (Cl(4)-cat)(Pyr)MoFe(3)S(3)(P(n)Pr(3))(3)(CO)(4) (VIII). In addition the new (Cl(4)-cat)(O)MoFe(3)S(3)(P(n)Pr(3))(3)(CO)(5) cluster (IVa), the (Cl(4)-cat)(O)MoFe(3)S(3)(PEt(3))(2)(CO)(6)cluster (V), the (Cl(4)-cat)(O)MoFe(3)S(3)(P(n)Pr(3))(2)(CO)(6) cluster (Va), the (Cl(4)-cat)(Pyr)MoFe(3)S(3)(P(n)Pr(3))(2)(CO)(6) cluster (VIa), and the (Cl(4)-cat)(P(n)Pr(3))MoFe(3)S(3)(P(n)Pr(3))(2)(CO)(6) cluster (VII) also are reported. Clusters III-VIII have been structurally and spectroscopically characterized. EPR, zero-field (57)Fe-Mössbauer spectroscopic characterizations, and magnetic susceptibility measurements have been used for a tentative assignment of the electronic and oxidation states of the MoFe(3)S(3) sulfur-voided cuboidal clusters. A structural comparison of the clusters with the MoFe(3)S(3) subunit of the FeMo-cofactor has led to the suggestion that the storage of reducing equivalents into M-M bonds, and their use in the reduction of substrates, may occur with the FeMo-cofactor, which also appears to have M-M bonding. On the basis of this argument, a possible N(2)-binding and reduction mechanism on the FeMoco-cofactor is proposed.

  18. Effect of adding cofactors to exogenous fibrolytic enzymes on preingestive hydrolysis, in vitro digestibility, and fermentation of bermudagrass haylage.

    Science.gov (United States)

    Romero, J J; Ma, Z X; Gonzalez, C F; Adesogan, A T

    2015-07-01

    Our objectives were to examine if adding metal ion cofactors (COF) to exogenous fibrolytic enzymes (EFE) would increase the beneficial effects of the EFE on the preingestive hydrolysis and in vitro digestibility and fermentation of bermudagrass haylage. In experiment 1, 5 COF (Mn(2+), Co(2+), Fe(2+), Ca(2+), and Mg(2+)) were screened to select the best candidates for synergistically enhancing release of water-soluble carbohydrates (WSC) from bermudagrass haylage by 5 EFE. The 5 EFE (1A, 2A, 11C, 13D, and 15D) were sourced from Trichoderma reesei and Aspergillus oryzae and they were the most effective of 12 EFE at increasing the neutral detergent fiber digestibility of bermudagrass haylage in a previous trial. Adding 1mM of each of the COF to EFE 2A or 11C synergistically increased release of WSC from bermudagrass haylage, as did adding (1mM) Fe(2+) to 1A, Mn(2+), Co(2+), or Fe(2+) to 13D, or Co(2+)or Fe(2+) to 15D. The greatest release of WSC responses were obtained by adding Mn(2+) to 11C (38%) or by adding Fe(2+) to 2A or 13D (10 and 21.9%, respectively). In experiment 2, the effect of increasing the COF dose on in vitro digestibility and fermentation of bermudagrass haylage was examined using the best EFE-COF combinations from experiment 1. Effects of adding increasing doses of these COF on EFE-mediated changes in vitro digestibility depended on the COF-EFE combination. Adding 10mM Mn(2+) alone to bermudagrass haylage increased DMD and NDFD by 2.7 and 6.3% and adding 11C alone increased these measures by 6.6 and 15.5%, respectively. However, adding 10mM Mn(2+) with 11C resulted in 3.5 and 8.1% increases in DMD and NDFD, respectively, beyond the increases caused by adding 11C alone. Adding Fe(2+) to 2A had no effects on EFE-mediated digestibility responses, but 2A prevented adverse effects of adding Fe(2+) alone on DMD and NDFD. In contrast, adding Fe(2+) to 13D reduced the increases in DMD and NDFD caused by adding the EFE alone. This study shows that adding COF

  19. Pa2G4 is a novel Six1 co-factor that is required for neural crest and otic development☆

    Science.gov (United States)

    Neilson, Karen M.; Abbruzzesse, Genevieve; Kenyon, Kristy; Bartolo, Vanessa; Krohn, Patrick; Alfandari, Dominique; Moody, Sally A.

    2016-01-01

    Mutations in SIX1 and in its co-factor, EYA1, underlie Branchiootorenal Spectrum disorder (BOS), which is characterized by variable branchial arch, otic and kidney malformations. However, mutations in these two genes are identified in only half of patients. We screened for other potential co-factors, and herein characterize one of them, Pa2G4 (aka Ebp1/Plfap). In human embryonic kidney cells, Pa2G4 binds to Six1 and interferes with the Six1-Eya1 complex. In Xenopus embryos, knock-down of Pa2G4 leads to down-regulation of neural border zone, neural crest and cranial placode genes, and concomitant expansion of neural plate genes. Gain-of-function leads to a broader neural border zone, expanded neural crest and altered cranial placode domains. In loss-of-function assays, the later developing otocyst is reduced in size, which impacts gene expression. In contrast, the size of the otocyst in gain-of-function assays is not changed but the expression domains of several otocyst genes are reduced. Together these findings establish an interaction between Pa2G4 and Six1, and demonstrate that it has an important role in the development of tissues affected in BOS. Thereby, we suggest that pa2g4 is a potential candidate gene for BOS. PMID:27940157

  20. Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase.

    Science.gov (United States)

    Riedel, B; Fiskerstrand, T; Refsum, H; Ueland, P M

    1999-07-01

    We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to ordinate distribution of Cbl cofactors during depletion and repletion.

  1. Prevalence of Anti Human Herpes Virus-6 IgG and its Receptor in Acute Leukemia (Membrane Cofactor Protein: MCP, CD46)

    International Nuclear Information System (INIS)

    Assem, M.M; El-Sharkawy, N.M.; Tarek, H.; Kamel, A.M.; Gad, W.H.; El-Rouby, M.N.; Ghaleb, F.M.

    2005-01-01

    CD46 is a membrane cofactor protein, which acts as a cofactor for factor I proteolytic cleavage of C3, so it protects the cells expressing it on their surface from autologous complement attack. It has been recently described as a receptor for HHV-6. Also, it has been shown to be highly expressed on malignant cells as compared to normal cells, thus playing a major role by which these cells, either cells of haematological malignancy or cells of other body cancers, can protect themselves against complement attack so they can survive and metastasize. Patients and methods: This study has been done to detect the sero prevalence of HHV-6 among 47 Egyptian adult cases of acute leukemia using the anti-HHV-6 IgG ELISA serological technique. CD46 receptor expression and immuno phenotyping technique were performed using FCM. Twenty nine of the cases were ANLL, while 18 were ALL cases. Sixteen age- and sex-matched control cases were also studied for both anti-HHV-6 IgG and CD46 receptor expression. HHV-6 IgG antibodies were encountered in 29 (100%), 14 (77.8%) and 12 (75%) of the ANLL, ALL and the control group, cases, respectively. CD46 expression was encountered in 21 (72.4%) of the ANLL cases and in 10 (55.6%) of the ALL cases. Concordance between HHV6 sero positivity and CD46 expression was encountered in 31 cases (29 positive and 2 negative). Dis concordance was encountered in 16 cases with 14 showing HHV-6 IgG sero positivity with no CD46 expression and 2 showing the reverse. The lack of significant correlation between CD46 expression and sero positivity would exclude CD46 expression as a cause of contracting HHV-6 infection in leukemic patients

  2. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

    International Nuclear Information System (INIS)

    Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

    2004-01-01

    Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

  3. The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

    Science.gov (United States)

    Fleischli, Christoph; Verhaagh, Sandra; Havenga, Menzo; Sirena, Dominique; Schaffner, Walter; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2005-08-01

    The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

  4. Assembling Fe/S-clusters and modifying tRNAs: ancient co-factors meet ancient adaptors.

    Science.gov (United States)

    Alfonzo, Juan D; Lukeš, Julius

    2011-06-01

    Trypanosoma brucei undergoes two clearly distinct develomental stages: in the insect vector (procyclic stage) the cells generate the bulk of their energy through respiration, whereas in the bloodstream of the mammalian host (bloodstream stage) they grow mostly glycolytically. Several mitochondrial respiratory proteins require iron-sulfur clusters for activity, and their activation coincides with developmental changes. Likewise some tRNA modification enzymes either require iron-sulfur clusters or use components of the iron-sulfur cluster assembly pathway for activity. These enzymes affect the anticodon loop of various tRNAs and can impact protein synthesis. Herein, the possibility of these pathways being integrated and exploited by T. brucei to carefully coordinate energy demands to translational rates in response to enviromental changes is examined.

  5. Efficient electrochemical regeneration of nicotinamide cofactors using a cyclopentadienyl-rhodium complex on functionalized indium tin oxide electrodes

    International Nuclear Information System (INIS)

    Kim, Soojin; Lee, Ga Ye; Lee, Jungha; Rajkumar, Eswaran; Baeg, Jin-Ook; Kim, Jinheung

    2013-01-01

    Functionalized ITO electrodes are used to regenerate NADH using [Cp*Rh(bpy)(H 2 O)] 2+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine) electrochemically in a buffer solution. Amino- and mercapto-functionalized electrodes featured higher activity and stability for electrocatalytic generation of NADH than a bare ITO electrode. Effect of metal nanoparticles was also studied on modified ITO electrodes and the addition of platinum nanoparticles even resulted in improved activity. The electrochemical regeneration was somewhat affected in the presence of dioxygen, but not significantly. In addition, a conversion of carbon dioxide was carried out utilizing the electrochemically generated NADH and formate dehydrogenase to produce formic acid

  6. Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

    Directory of Open Access Journals (Sweden)

    Alexey Navdaev

    Full Text Available von Willebrand factor/ristocetin (vWF/R induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

  7. Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

    Science.gov (United States)

    Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

    2014-01-01

    von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

  8. Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

    Science.gov (United States)

    Florea, Mara; Nau, Werner M

    2010-03-07

    A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

  9. A Novel Nonsense Variant in Nav1.5 Cofactor MOG1 Eliminates Its Sodium Current Increasing Effect and May Increase the Risk of Arrhythmias

    DEFF Research Database (Denmark)

    Olesen, Morten S; Jensen, Niels F; Holst, Anders G

    2011-01-01

    at a lower frequency (1.8% vs 0.4%, P = 0.078). Electrophysiological investigation showed that the p.E61X variant completely eliminates the sodium current-increasing effect of MOG1 and thereby causes loss of function in the sodium current. When mimicking heterozygosity by coexpression of Nav1.5 with wild......BACKGROUND: The protein MOG1 is a cofactor of the cardiac sodium channel, Nav1.5. Overexpression of MOG1 in Nav1.5-expressing cells increases sodium current markedly. Mutations in the genes encoding Nav1.5 and its accessory proteins have been associated with cardiac arrhythmias of significant...... and 23 were patients with Brugada syndrome. The effect of one variant was investigated functionally by patch-clamping CHO-K1 cells coexpressing Nav1.5 with MOG1. RESULTS: We uncovered a novel heterozygous nonsense variant, c.181G>T (p.E61X), that, however, was also present in control subjects, albeit...

  10. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

    KAUST Repository

    Schmeier, Sebastian

    2016-10-17

    Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

  11. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

    KAUST Repository

    Schmeier, Sebastian; Alam, Tanvir; Essack, Magbubah; Bajic, Vladimir B.

    2016-01-01

    Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

  12. The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

    International Nuclear Information System (INIS)

    Kowalski, K.; Czolij, R.; King, G.F.; Crossley, M.; Mackay, J.P.

    1999-01-01

    Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted β-hairpins and a single α-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA- binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions

  13. Estimating HIV Incidence during Pregnancy and Knowledge of Prevention of Mother-to-Child Transmission with an Ad Hoc Analysis of Potential Cofactors

    Directory of Open Access Journals (Sweden)

    Thomas Obinchemti Egbe

    2016-01-01

    Full Text Available Background. We determined the incidence of HIV seroconversion during the second and third trimesters of pregnancy and ad hoc potential cofactors associated with HIV seroconversion after having an HIV-negative result antenatally. We also studied knowledge of PMTCT among pregnant women in seven health facilities in Fako Division, South West Region, Cameroon. Method. During the period between September 12 and December 4, 2011, we recruited a cohort of 477 HIV-negative pregnant women by cluster sampling. Data collection was with a pretested interviewer-administered questionnaire. Sociodemographic information, knowledge of PMTCT, and methods of HIV prevention were obtained from the study population and we did Voluntary Counselling and Testing (VCT for HIV. Results. The incidence rate of HIV seroconversion during pregnancy was 6.8/100 woman-years. Ninety percent of the participants did not use condoms throughout pregnancy but had a good knowledge of PMTCT of HIV. Only 31.9% of participants knew their HIV status before the booking visit and 33% did not know the HIV status of their partners. Conclusion. The incidence rate of HIV seroconversion in the Fako Division, Cameroon, was 6.8/100 woman-years. No risk factors associated with HIV seroconversion were identified among the study participants because of lack of power to do so.

  14. Estimating HIV Incidence during Pregnancy and Knowledge of Prevention of Mother-to-Child Transmission with an Ad Hoc Analysis of Potential Cofactors.

    Science.gov (United States)

    Egbe, Thomas Obinchemti; Tazinya, Rose-Mary Asong; Halle-Ekane, Gregory Edie; Egbe, Eta-Nkongho; Achidi, Eric Akum

    2016-01-01

    We determined the incidence of HIV seroconversion during the second and third trimesters of pregnancy and ad hoc potential cofactors associated with HIV seroconversion after having an HIV-negative result antenatally. We also studied knowledge of PMTCT among pregnant women in seven health facilities in Fako Division, South West Region, Cameroon. During the period between September 12 and December 4, 2011, we recruited a cohort of 477 HIV-negative pregnant women by cluster sampling. Data collection was with a pretested interviewer-administered questionnaire. Sociodemographic information, knowledge of PMTCT, and methods of HIV prevention were obtained from the study population and we did Voluntary Counselling and Testing (VCT) for HIV. The incidence rate of HIV seroconversion during pregnancy was 6.8/100 woman-years. Ninety percent of the participants did not use condoms throughout pregnancy but had a good knowledge of PMTCT of HIV. Only 31.9% of participants knew their HIV status before the booking visit and 33% did not know the HIV status of their partners. The incidence rate of HIV seroconversion in the Fako Division, Cameroon, was 6.8/100 woman-years. No risk factors associated with HIV seroconversion were identified among the study participants because of lack of power to do so.

  15. The Crystal Structure of Cobra Venom Factor, a Cofactor for C3- and C5-Convertase CVFBb

    Energy Technology Data Exchange (ETDEWEB)

    Krishnan, Vengadesan; Ponnuraj, Karthe; Xu, Yuanyuan; Macon, Kevin; Volanakis, John E.; Narayana, Sthanam V.L.; (Madras); (UAB)

    2009-05-26

    Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 {angstrom} resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.

  16. Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor.

    Science.gov (United States)

    Vidal, Juan-C; Espuelas, Javier; Castillo, Juan-R

    2004-10-01

    A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.

  17. Six hours of resting platelet concentrates stored at 22-24 ºC for 48 hours in permeable bags preserved pH, swirling and lactate dehydrogenase better and caused less platelet activation.

    Science.gov (United States)

    Naghadeh, Hossin T; Badlou, Bahram A; Ferizhandy, Ali S; Mohammadreza, Tabatabai S; Shahram, Vaeli

    2013-07-01

    During transportation, platelet concentrates (PC) usually undergo a long period without agitation. Whether this interruption improves quality and viability or, contrariwise, has deleterious effects on PC stored for 48 hours (h) is unknown. The aim of this study was to investigate the effects of metabolic resting (6 h of interruption of agitation) vs continue agitation of PC stored for 48 h in the blood bank of Tehran. PC were prepared from platelet-rich plasma and stored in permeable bags in a shaker/incubator for 42 h at room temperature (20-24 ºC). Then, simply by stopping the agitator, the PC remained stationary ("resting") without agitation for 6 h (WCA6h), prior to transfusion. In vitro measurements of platelet quality were carried out just after completion of the resting period and the results were compared with those of PC continuously agitated in the same day (designated as the control group, CA6h). The in vitro variables measured were swirling, ristocetin-induced aggregation (GPIb-related function), lactate dehydrogenase (LDH) concentration, platelet factor 4 (PF4) release and P-selectin expression (activation markers). The mean platelet counts of the control group (CA6h) and rested (WCA6h) PC were not statistically different (P =0.548). Likewise, the mean pH values were not significantly different: WCA6h (7.16 ± 0.08) and CA6h (7.22 ± 0.16) (P =0.300). Although ristocetin-induced aggregation did not differ significantly between CA6h (79.2 ± 4.4) and WCA6h (66.65 ± 28.55) (P =0.186), WCA6h showed significantly less PFA release (P =0.015) and lower P-selectin expression (P =0.006). We observed that PC stored under agitation for 42 h at 22-24 ºC in permeable bags and then rested for 6 h had better preserved pH, swirling and LDH and less platelet activation then PC kept under continuous agitation for the whole 48 h storage period.

  18. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  19. Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

    Directory of Open Access Journals (Sweden)

    Cristina Puy

    Full Text Available Factor (F XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα, in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin.Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma.Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

  20. DNA methylation potential: dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women123

    Science.gov (United States)

    Dominguez-Salas, Paula; Moore, Sophie E; Cole, Darren; da Costa, Kerry-Ann; Cox, Sharon E; Dyer, Roger A; Fulford, Anthony JC; Innis, Sheila M; Waterland, Robert A; Zeisel, Steven H; Prentice, Andrew M; Hennig, Branwen J

    2013-01-01

    Background: Animal models show that periconceptional supplementation with folic acid, vitamin B-12, choline, and betaine can induce differences in offspring phenotype mediated by epigenetic changes in DNA. In humans, altered DNA methylation patterns have been observed in offspring whose mothers were exposed to famine or who conceived in the Gambian rainy season. Objective: The objective was to understand the seasonality of DNA methylation patterns in rural Gambian women. We studied natural variations in dietary intake of nutrients involved in methyl-donor pathways and their effect on the respective metabolic biomarkers. Design: In 30 women of reproductive age (18–45 y), we monitored diets monthly for 1 y by using 48-h weighed records to measure intakes of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12. Blood biomarkers of these nutrients, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), homocysteine, cysteine, and dimethylglycine were also assessed monthly. Results: Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season. Conclusions: Naturally occurring seasonal variations in food-consumption patterns have a profound effect on methyl-donor biomarker status. The direction of these changes was consistent with previously reported differences in methylation of metastable epialleles. This trial was registered at www.clinicaltrials.gov as NCT01811641. PMID:23576045

  1. The transcriptional co-factor RIP140 regulates mammary gland development by promoting the generation of key mitogenic signals.

    Science.gov (United States)

    Nautiyal, Jaya; Steel, Jennifer H; Mane, Meritxell Rosell; Oduwole, Olayiwola; Poliandri, Ariel; Alexi, Xanthippi; Wood, Nicholas; Poutanen, Matti; Zwart, Wilbert; Stingl, John; Parker, Malcolm G

    2013-03-01

    Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.

  2. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

    Science.gov (United States)

    Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

    2014-02-20

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

  3. La mitocondria como fábrica de cofactores: biosíntesis de grupo hemo, centros Fe-S y nucleótidos de flavina (FMN/FAD)

    OpenAIRE

    Alexa Villavicencio-Queijeiro

    2012-01-01

    Los cofactores hemo, centros Fe-S y los nucleótidos de flavina (FMN y FAD) son esenciales para muchos organismos, existen un gran número de proteínas que dependen de ellos para llevar a cabo sus funciones biológicas. Estos cofactores han sido reconocidos como esenciales para las reacciones de óxido-reducción, pero también están involucrados en otros procesos celulares como la catálisis química, la regulación, la señalización y la detección de señales intra y extra celulares. Diversos grupos d...

  4. NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease.

    Directory of Open Access Journals (Sweden)

    Xun-Cheng Su

    Full Text Available BACKGROUND: The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation in the absence of inhibitor and lining the substrate binding site (closed conformation in the presence of an inhibitor. METHODS: In this work, nuclear magnetic resonance (NMR spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution. FINDINGS: In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors. CONCLUSION: Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.

  5. La mitocondria como fábrica de cofactores: biosíntesis de grupo hemo, centros Fe-S y nucleótidos de flavina (FMN/FAD

    Directory of Open Access Journals (Sweden)

    Alexa Villavicencio-Queijeiro

    2012-01-01

    Full Text Available Los cofactores hemo, centros Fe-S y los nucleótidos de flavina (FMN y FAD son esenciales para muchos organismos, existen un gran número de proteínas que dependen de ellos para llevar a cabo sus funciones biológicas. Estos cofactores han sido reconocidos como esenciales para las reacciones de óxido-reducción, pero también están involucrados en otros procesos celulares como la catálisis química, la regulación, la señalización y la detección de señales intra y extra celulares. Diversos grupos de investigación han contribuido al establecimiento de las rutas bioquímicas por las que se sintetizan estos cofactores, así como a la forma en que se transportan y regulan en los diferentes organismos. Todo este conocimiento ha permitido asociar algunas enfermedades con defectos metabólicos en estas rutas de biosíntesis, así como plantear nuevas estrategias terapéuticas y algunas aplicaciones biotecnológicas.

  6. The Transcription Cofactor Swi6 of the Fusarium graminearum Is Involved in Fusarium Graminearum Virus 1 Infection-Induced Phenotypic Alterations

    Directory of Open Access Journals (Sweden)

    Moonil Son

    2016-08-01

    Full Text Available The transcription cofactor Swi6 plays important roles in regulating vegetative growth and meiosis in Saccharomyces cerevisiae. Functions of Swi6 ortholog were also characterized in Fusarium graminearum which is one of the devastating plant pathogenic fungi. Here, we report possible role of FgSwi6 in the interaction between F. graminearum and Fusarium graminearum virus 1 (FgV1 strain DK21. FgV1 perturbs biological characteristics of host fungi such as vegetative growth, sporulation, pigmentation, and reduction of the virulence (hypovirulence of its fungal host. To characterize function(s of FgSWI6 gene during FgV1 infection, targeted deletion, over-expression, and complementation mutants were generated and further infected successfully with FgV1. Deletion of FgSwi6 led to severe reduction of vegetative growth even aerial mycelia while over-expression did not affect any remarkable alteration of phenotype in virus-free isolates. Virus-infected (VI FgSWI6 deletion isolate exhibited completely delayed vegetative growth. However, VI FgSWI6 over-expression mutant grew faster than any other VI isolates. To verify whether these different growth patterns in VI isolates, viral RNA quantification was carried out using qRT-PCR. Surprisingly, viral RNA accumulations in VI isolates were similar regardless of introduced mutations. These results provide evidence that FgSWI6 might play important role(s in FgV1 induced phenotype alteration such as delayed vegetative growth.

  7. Catch a tiger snake by its tail: Differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes.

    Science.gov (United States)

    Lister, Callum; Arbuckle, Kevin; Jackson, Timothy N W; Debono, Jordan; Zdenek, Christina N; Dashevsky, Daniel; Dunstan, Nathan; Allen, Luke; Hay, Chris; Bush, Brian; Gillett, Amber; Fry, Bryan G

    2017-11-01

    A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

    Science.gov (United States)

    Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2007-11-01

    We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

  9. Cofactors in the RNA World

    Science.gov (United States)

    Ditzler, Mark A.

    2014-01-01

    RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

  10. Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic

  11. Determination of coagulation inhibitor levels and resistance to activated protein C in patients undergoing gastric surgery for benign and malignant disorders

    DEFF Research Database (Denmark)

    Andersen, B S; Rahr, H B; Sørensen, J V

    1997-01-01

    The aim of the present study was to determine plasma levels of protein C antigen (PC:Ag) and activity (PC:Act), tissue factor pathway inhibitor (TFPI), protein S (PS), antithrombin (AT), heparin cofactor II (HCII), and resistance to activated protein C (APCR) before, during and after elective gas...

  12. Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

    Science.gov (United States)

    Papapetridis, Ioannis; van Dijk, Marlous; Dobbe, Arthur P A; Metz, Benjamin; Pronk, Jack T; van Maris, Antonius J A

    2016-04-26

    Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor

  13. The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity.

    Science.gov (United States)

    Kiehler, Brittany; Haggett, Lindsey; Fujita, Masaya

    2017-08-01

    Sporulation in Bacillus subtilis is induced upon starvation. In a widely accepted model, an N-terminal "sensor" domain of the major sporulation kinase KinA recognizes a hypothetical starvation signal(s) and autophosphorylates a histidine residue to activate the master regulator Spo0A via a multicomponent phosphorelay. However, to date no confirmed signal has been found. Here, we demonstrated that PAS-A, the most N-terminal of the three PAS domains (PAS-ABC), is dispensable for the activity, contrary to a previous report. Our data indicated that the autokinase activity is dependent on the formation of a functional tetramer, which is mediated by, at least, PAS-B and PAS-C. Additionally, we ruled out the previously proposed notion that NAD + /NADH ratio controls KinA activity through the PAS-A domain by demonstrating that the cofactors show no effects on the kinase activity in vitro. In support of these data, we found that the cofactors exist in approximately 1000-fold excess of KinA in the cell and the cofactors' ratio does not change significantly during growth and sporulation, suggesting that changes in the cofactor ratio might not play a role in controlling KinA activity. These data may refute the widely-held belief that the activity of KinA is regulated in response to an unknown starvation signal(s). © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  14. Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements

    DEFF Research Database (Denmark)

    Blouse, Grant E; Dupont, Daniel Miotto; Schar, Christine R

    2009-01-01

    In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments de...

  15. Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

    Science.gov (United States)

    Carpentier, Jean; Grenier, Eric; Esquibet, Magalie; Hamel, Louis-Philippe; Moffett, Peter; Manzanares-Dauleux, Maria J; Kerlan, Marie-Claire

    2013-04-19

    The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene

  16. Structure and biological activity of a fucosylated chondroitin sulfate from the sea cucumber Cucumaria japonica.

    Science.gov (United States)

    Ustyuzhanina, Nadezhda E; Bilan, Maria I; Dmitrenok, Andrey S; Shashkov, Alexander S; Kusaykin, Mikhail I; Stonik, Valentin A; Nifantiev, Nikolay E; Usov, Anatolii I

    2016-05-01

    A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonicaby extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core → 3)-β-D-GalNAc-(1 → 4)-β-D-GlcA-(1 →. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, ∼ 33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence von Willebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Spectroscopic investigations of the B12-binding subunit of glutamate mutase: refined solution structure of the complex with the B12-nucleotide, dynamics and binding studies with two corrinoid cofactors

    International Nuclear Information System (INIS)

    Eichmueller, C.

    2002-06-01

    Glutamate mutase is an enzyme isolated from Clostridium tetanomorphum and Clostridium cochlearum. It catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. Coenzyme B12 is required as cofactor for an active enzyme, as the first step of the catalytic cycle is the homolytic cleavage of the cobalt-carbon bond. The rearrangement itself follows a radical mechanism. The holoenzyme is an alpha2beta2 heterotetramer containing two identical catalytic and two B12 binding domains, as well as two coenzyme B12 molecules. The smaller B12 binding domain from Clostridium tetanomorphum, MutS, is known to bind coenzyme B12 in its unusual 'base-off' form. A conserved histidine residue coordinates to the cobalt atom instead of the normally coordinated dimethlybenzimidole in free coenzyme B12. In the present work a refined solution structure of the B12 binding subunit from Clostridium tetanomorphum (MutS) in complex with the detached nucleotide loop of coenzyme B12 has been determined using nuclear magnetic resonance. The found topology is almost identical to the crystal structure of glutamate mutase from C.cochlearum [Reitzer et al., 1999], in contrast to the solution structures obtained for apo-MutS [Hoffmann et al., 2001; Tollinger et al., 1998] and apo-GlmS [Hoffmann et al., 1999]. In these two structures a helix at one side of the B12 nucleotide loop binding pocket is mostly unstructured and shows motions on a microsecond to millisecond timescale. The previously found stabilization of this helix upon B12-nucleotide binding [Tollinger et al., 2001] was confirmed using 13C and 15N labeled MutS. Some differences are found in the structure of the binding pocket and the bound nucleotide loop compared to the crystal structure. This indicates that additional conformational changes occur upon binding of the corrin ring of coenzyme B12. NMR-relaxation measurements performed on apo-MutS showed interesting slow molecular motions not only in the mainly

  18. Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

    Science.gov (United States)

    Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

    2017-01-05

    BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5A In Vitro

    Directory of Open Access Journals (Sweden)

    Ankush Gupta

    2014-01-01

    Full Text Available Ribonuclease L (RNase L is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

  20. Curcumin, a natural antioxidant, acts as a noncompetitive inhibitor of human RNase L in presence of its cofactor 2-5A in vitro.

    Science.gov (United States)

    Gupta, Ankush; Rath, Pramod C

    2014-01-01

    Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

  1. An empirical model of Onecut binding activity at the sea urchin SM50 C-element gene regulatory region.

    Science.gov (United States)

    Otim, Ochan

    2017-01-01

    Studying the formation of endoskeleton in many species is complex and difficult. The sea urchin embryo offers an unparalleled platform for understanding this process because of the ease with which its skeletogenic mesenchyme cells can be manipulated. In this study, preliminary evidence from biochemical studies towards understanding the role of the Onecut transcription factor during sea urchin skeletogenic mesenchyme cell specification is presented. Based on the evidence, an empirical model is proposed showing how Onecut, together with associated co-factors, may be using the C-element of the SM50 gene regulatory region in advance of the sea urchin Strongylocentrotus purpuratus spicule development. In the model, Onecut recognizes and binds the DNA sequence CATCGATCTC in the C-element without temporal restriction. Onecut then utilizes different sets of co-factors to switch from its unknown function early in development (four cell stage to the mesenchyme blastula stage), to its known role in the oral-aboral boundary thereafter. At the writing of this report, definitive evidence as to whether the "early" factors are expressed in all cells except the micromere lineages, or whether the "late" factors are expressed in micromere descendants or ectodermal precursors only are lacking. The former would suggest a possible Onecut repression function for the early co-factors outside the micromere lineages; the latter scenario would suggest a Onecut activation function for the late co-factors in the presumptive ciliary band.

  2. Studies of the variability of the hepatocyte nuclear factor-1beta (HNF-1beta / TCF2) and the dimerization cofactor of HNF-1 (DcoH / PCBD) genes in relation to type 2 diabetes mellitus and beta-cell function

    DEFF Research Database (Denmark)

    Ek, J; Grarup, N; Urhammer, S A

    2001-01-01

    Mutations in the homeodomain-containing transcription factor hepatocyte nuclear factor-1beta (HNF-1beta) are known to cause a rare subtype of maturity-onset diabetes of the young (MODY5), which is associated with early-onset progressive non-diabetic renal dysfunction. To investigate whether...... mutations in HNF-1 are implicated in the pathogenesis of MODY or late-onset diabetes with and without nephropathy in Danish Caucasians we examined the HNF-1beta (TCF2) and the dimerization cofactor of HNF-1 (DCoH, PCBD) genes for mutations in 11 MODY probands, 28 type 2 diabetic patients with nephropathy...... comprising the DCoH gene revealed a previously described A-->G polymorphism located in the 3' untranslated region, which was not investigated further. In conclusion, mutations in HNF-1beta and DCoH are not a major cause of MODY or late onset type 2 diabetes in Danish Caucasian subjects....

  3. Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet.

    Directory of Open Access Journals (Sweden)

    Donald R Senger

    Full Text Available The Nrf2 (NFE2L2 cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both in vitro and in vivo. Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The

  4. Manganese effect over the bacteria activity on the active mires process; Efecto del Manganeso sobre la actividad bacteriana en procesos de fangos activos

    Energy Technology Data Exchange (ETDEWEB)

    Simon Andreu, P. J.; Lardin Mifsud, C.; Martinez Muro, M. A.; Vicente Gonzalez, J. A.; Sanchez Beltran, A. V.; Gonzalez Herrero, R.; Llosa Llacer, C.

    2009-07-01

    With this assay, it has been determined the incidence of manganese (Mn (II) in the metabolic activity of bacteria involved in biological processes in a WWTP. It has been observed the function of Mn (II) as an enzymatic co-factor and its influence on the increase of bacteria oxidative capacity. Besides, it has been studied the effect of Mn (II) as a metabolic uncoupler and its influence on biomass production decreases. (Author)

  5. High prevalence of sensitization to gibberellin-regulated protein (peamaclein) in fruit allergies with negative immunoglobulin E reactivity to Bet v 1 homologs and profilin: Clinical pattern, causative fruits and cofactor effect of gibberellin-regulated protein allergy.

    Science.gov (United States)

    Inomata, Naoko; Miyakawa, Mami; Aihara, Michiko

    2017-07-01

    Gibberellin-regulated protein (GRP) is a new allergen in peach allergy, with an amino acid sequence very well conserved through several botanical species. We investigated the allergenicity of GRP in fruit allergies other than peaches and identified the clinical characteristics of fruit allergy patients with GRP sensitization. One hundred consecutive Japanese patients with fruit allergies were enrolled in the present study. To identify the features of GRP sensitization, we selected patients with negative ImmunoCAP results for Bet v 1 homologs and profilin, which are marker allergens for pollen-food allergy syndrome (PFAS), or lipid transfer protein. These patients underwent specific immunoglobulin E measurements by enzyme-linked immunosorbent assay (ELISA) and skin prick tests (SPT) using purified nPru p 7. Twenty of 100 consecutive patients with fruit allergies had negative ImmunoCAP results for Bet v 1 homologs and profilin. Thirteen (65.0%) of the 20 patients had positive ELISA and/or SPT results using nPru p 7, whereas one of the 20 patients had positive ImmunoCAP results for Pru p 3. In 13 nPru p 7-sensitized patients, the causative foods were peaches (92.3%), apricots (61.5%), oranges (46.2%) and apples (30.8%). Ten patients (76.9%) had multiple causative fruits. Frequent symptoms included facial edema (92.3%) and laryngeal tightness (66.7%). In eight patients (61.5%), exercise or aspirin intake enhanced the allergic reaction onset as cofactors. The prevalence of GRP sensitization was high in Japanese fruit allergy patients except for PFAS patients. In conclusion, GRP-sensitized patients may have allergies to multiple fruits and may show peculiar characteristics such as facial swelling and cofactor dependence. © 2017 Japanese Dermatological Association.

  6. The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways

    NARCIS (Netherlands)

    Conway, Edward M.; van de Wouwer, Marlies; Pollefeyt, Saskia; Jurk, Kerstin; van Aken, Hugo; de Vriese, Astrid; Weitz, Jeffrey I.; Weiler, Hartmut; Hellings, Peter W.; Schaeffer, Paul; Herbert, Jean-Marc; Collen, Désiré; Theilmeier, Gregor

    2002-01-01

    Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH(2)-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack

  7. Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor

    NARCIS (Netherlands)

    W. Klootwijk (Willem); T.J. Visser (Theo); G.G.J.M. Kuiper (George)

    2002-01-01

    textabstractHuman type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all

  8. Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A.

    Science.gov (United States)

    O'Brien, D P; Tuddenham, E G

    1989-06-01

    We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

  9. Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

    OpenAIRE

    Wu, Shwu-Yuan; Zhou, Tianyuan; Chiang, Cheng-Ming

    2003-01-01

    Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by e...

  10. Biosynthesis of the Essential Fatty Acid Oxidation Cofactor Carnitine Is Stimulated in Heart and Liver after a Single Bout of Exercise in Mice

    Directory of Open Access Journals (Sweden)

    Tom L. Broderick

    2018-01-01

    Full Text Available We determined whether one single bout of exercise stimulates carnitine biosynthesis and carnitine uptake in liver and heart. Free carnitine (FC in plasma was assayed using acetyltransferase and [14C]acetyl-CoA in Swiss Webster mice after 1 hour of moderate-intensity treadmill running or 4 hours and 8 hours into recovery. Liver and heart were removed under the same conditions for measurement of carnitine biosynthesis enzymes (liver butyrobetaine hydroxylase, γ-BBH; heart trimethyllysine dioxygenase, TMLD, organic cation transporter-2 (OCTN2, carnitine transporter, and liver peroxisome proliferator-activated receptor-alpha (PPARα, transcription factor for γ-BBH and OCTN2 synthesis. In exercised mice, FC levels in plasma decreased while heart and liver OCTN2 protein expressed increased, reflecting active uptake of FC. During recovery, the rise in FC to control levels was associated with increased liver γ-BBH expression. Protein expression of PPARα was stimulated in liver after exercise and during recovery. Interestingly, heart TMLD protein was also detected after exercise. Acute exercise stimulates carnitine uptake in liver and heart. The rapid return of FC levels in plasma after exercise indicates carnitine biosynthesis by liver is stimulated to establish carnitine homeostasis. Our results suggest that exercise may benefit patients with carnitine deficiency syndromes.

  11. Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings ( Pisum sativum) and Arthrobacter globiformis: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor.

    Science.gov (United States)

    Shepard, Eric M; Juda, Gregory A; Ling, Ke-Qing; Sayre, Lawrence M; Dooley, David M

    2004-04-01

    The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ(amr)-Cu(II) right harpoon over left harpoon TPQ(sq)-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O(2) for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K(d) values for Cu(II)-CN(-) and Cu(I)-CN(-), as well as the K(i) for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN(-) complexation of Cu(I).

  12. Article Commentary: Kynurenine Pathway Pathologies: Do Nicotinamide and Other Pathway Co-Factors have a Therapeutic Role in Reduction of Symptom Severity, Including Chronic Fatigue Syndrome (CFS and Fibromyalgia (FM

    Directory of Open Access Journals (Sweden)

    Adele Blankfield

    2013-01-01

    Full Text Available Chronic fatigue syndrome (CFS and fibromyalgia (FM appear to meet the criteria of a tryptophan-kynurenine pathway disorder with potential neuroimmunological sequelae. Aspects of some of the putative precipitating factors have been previously outlined. 2 , 3 An analysis of the areas of metabolic dysfunction will focus on future directions for research and management. The definition of dual tryptophan pathways has increased the understanding of the mind-body, body-mind dichotomy. The serotonergic pathway highlights the primary (endogenous psychiatric disorders. The up-regulation of the kynurenine pathway by physical illnesses can cause neuropathic and immunological disorders 1 associated with secondary neuropsychiatric symptoms. Tryptophan and nicotinamide deficiencies fall within the protein energy malnutrition (PEM spectrum. They can arise if the kynurenine pathway is stressed by primary or secondary inflammatory conditions and the consequent imbalance of available catabolic/anabolic substrates may adversely influence convalescent phase efficiency. The replacement of depleted or reduced NAD+ levels and other cofactors can perhaps improve the clinical management of these disorders.

  13. Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

    Science.gov (United States)

    Nagatsu, Toshiharu; Nagatsu, Ikuko

    2016-11-01

    Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

  14. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    International Nuclear Information System (INIS)

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P.

    1989-01-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125 I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

  15. Replacing Electron Transport Cofactors with Hydrogenases

    KAUST Repository

    Laamarti, Rkia

    2016-01-01

    to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a

  16. Cofactors Influencing Prevalence and Intensity of Schistosoma ...

    African Journals Online (AJOL)

    Urine samples were collected from 657 individuals and analyzed by centrifugation, and the number of ova was determined by microscopy. ... Cattle rearing (OR=9.01; CI=4.00-20.75; P=0.00) and farming (OR=3.14; CI=1.82-5.43; P=0.00) showed significant association with the prevalence and intensity of the disease.

  17. COFACTORS INFLUENCING PREVALENCE AND INTENSITY OF ...

    African Journals Online (AJOL)

    Administrator

    Urine samples were collected from 657 individuals and analyzed by centrifugation, and the number of ova was determined by microscopy. The population had an .... infection/10ml urine. Odd Ratio. (95%C.I). Chi-Square. (χ2). P-value. Herdsmen. 34. 24. 70.59. 160.42±76.87. 9.01(4.00,20.75). 41.23. 0.0000*. Farmers. 67.

  18. Potent antitumor activity of a urokinase-activated engineered anthrax toxin

    Science.gov (United States)

    Liu, Shihui; Aaronson, Hannah; Mitola, David J.; Leppla, Stephen H.; Bugge, Thomas H.

    2003-01-01

    The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

  19. Von Willebrand's disease: case report and review of literature.

    Science.gov (United States)

    Echahdi, Hanae; El Hasbaoui, Brahim; El Khorassani, Mohamed; Agadr, Aomar; Khattab, Mohamed

    2017-01-01

    Von Willebrand Disease (VWD) is the most common human inherited bleeding disorder due to a defect of Von Willebrand Factor (VWF), which a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels < 50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50IU/dL). Von willebrand factor is a complex multimeric protein with two functions: it forms a bridge between the platelets and areas of vascular damage and it binds to and stabilizes factor VIII, which is necessary for the clotting cascade. By taking a clinical history of bleeding (mucocutaneous bleeding symptoms suggestive of a primary haemostatic disorder, a quantitative or qualitative abnormality of VWF is possible) it is important to think about VWD and to make the appropriate diagnosis. If the VWD is suspected diagnostic tests should include an activated partial thromboplastin time, bleeding time, factor VIII: C Ristocetin cofactor and vWF antigen. Additional testing of ristocetin induced plattlet adhesion (RIPA) the multimeric structure and collagen binding test and genanalysis allow diagnosing the different types of von. Willebrand Disease. The treatment of choice in mild forms is the synthetic agent desmopressin. In patients with severe type 1, type 2B, 2N and type 3 or in people who do not response to desmopressin, the appropriate treatment is a factor VIII concentrate that is rich of VWF. We report a case of infant in 27-month-old boy who had been referred due to haemorrhagic shock. His birth histories, his familie's social history and developmental milestones were unremarkable. He was born at full term with no antenatal or perinatal complications. Prior to

  20. Long-Lived Termite Queens Exhibit High Cu/Zn-Superoxide Dismutase Activity

    Directory of Open Access Journals (Sweden)

    Eisuke Tasaki

    2018-01-01

    Full Text Available In most organisms, superoxide dismutases (SODs are among the most effective antioxidant enzymes that regulate the reactive oxygen species (ROS generated by oxidative energy metabolism. ROS are considered main proximate causes of aging. However, it remains unclear if SOD activities are associated with organismal longevity. The queens of eusocial insects, such as termites, ants, and honeybees, exhibit extraordinary longevity in comparison with the nonreproductive castes, such as workers. Therefore, the queens are promising candidates to study the underlying mechanisms of aging. Here, we found that queens have higher Cu/Zn-SOD activity than nonreproductive individuals of the termite Reticulitermes speratus. We identified three Cu/Zn-SOD sequences and one Mn-SOD sequence by RNA sequencing in R. speratus. Although the queens showed higher Cu/Zn-SOD activity than the nonreproductive individuals, there were no differences in their expression levels of the Cu/Zn-SOD genes RsSOD1 and RsSOD3A. Copper (Cu2+ and Cu+ is an essential cofactor for Cu/Zn-SOD enzyme activity, and the queens had higher concentrations of copper than the workers. These results suggest that the high Cu/Zn-SOD activity of termite queens is related to their high levels of the cofactor rather than gene expression. This study highlights that Cu/Zn-SOD activity contributes to extraordinary longevity in termites.

  1. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  2. A role of active brown adipose tissue in cancer cachexia?

    Directory of Open Access Journals (Sweden)

    Emiel Beijer

    2012-06-01

    Full Text Available Until a few years ago, adult humans were not thought to have brown adipose tissue (BAT. Now, this is a rapidly evolving field of research with perspectives in metabolic syndromes such as obesity and new therapies targeting its bio-energetic pathways. White, brown and socalled brite adipose fat seem to be able to trans-differentiate into each other, emphasizing the dynamic nature of fat tissue for metabolism. Human and animal data in cancer cachexia to date provide some evidence for BAT activation, but its quantitative impact on energy expenditure and weight loss is controversial. Prospective clinical studies can address the potential role of BAT in cancer cachexia using 18F-fluorodeoxyglucose positron emission tomography-computed tomography scanning, with careful consideration of co-factors such as diet, exposure to the cold, physical activity and body mass index, that all seem to act on BAT recruitment and activity.

  3. Spectroscopic and computational studies of cobalamin species with variable lower axial ligation: implications for the mechanism of Co-C bond activation by class I cobalamin-dependent isomerases.

    Science.gov (United States)

    Conrad, Karen S; Jordan, Christopher D; Brown, Kenneth L; Brunold, Thomas C

    2015-04-20

    5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor's Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor's electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.

  4. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  5. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  6. Dietary factor VII activation does not increase plasma concentrations of prothrombin fragment 1+2 in patients with stable angina pectoris and coronary atherosclerosis

    DEFF Research Database (Denmark)

    Bladbjerg, E-M; Münster, A M; Marckmann, P

    2000-01-01

    Studies in healthy subjects showed that blood coagulation factor VII (FVII) is activated postprandially after consumption of high-fat meals, but accompanying thrombin formation has not been demonstrated. In patients with coronary atherosclerosis, the arterial intima is supposed to present more...... tissue factor, the cofactor of FVII, to circulating blood; therefore, thrombin formation in response to FVII activation is more likely to occur in such patients. This hypothesis was tested in a randomized crossover study of 30 patients (aged 43 to 70 years) with stable angina pectoris...

  7. Overview of platelet physiology and laboratory evaluation of platelet function.

    Science.gov (United States)

    Rodgers, G M

    1999-06-01

    Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays

  8. Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

    Science.gov (United States)

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

  9. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  10. Experimentally calibrated computational chemistry of tryptophan hydroxylase: Trans influence, hydrogen-bonding, and 18-electron rule govern O-2-activation

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink; Kepp, Kasper Planeta; Boesen, Jane

    2010-01-01

    with the experimental value (0.25 mm/s) which we propose as the structure of the hydroxylating intermediate, with the tryptophan substrate well located for further reaction 3.5 Å from the ferryl group. Based on the optimized transition states, the activation barriers for the two paths (glu and his) are similar, so......Insight into the nature of oxygen activation in tryptophan hydroxylase has been obtained from density functional computations. Conformations of O2-bound intermediates have been studied with oxygen trans to glutamate and histidine, respectively. An O2-adduct with O2 trans to histidine (Ohis...... towards the cofactor and a more activated O–O bond (1.33 Å) than in Oglu (1.30 Å). It is shown that the cofactor can hydrogen bond to O2 and activate the O–O bond further (from 1.33 to 1.38 Å). The Ohis intermediate leads to a ferryl intermediate (Fhis) with an isomer shift of 0.34 mm/s, also consistent...

  11. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

    DEFF Research Database (Denmark)

    Hallenborg, P.; Siersbæk, M.; Barrio-Hernandez, I.

    2016-01-01

    on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each......The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies...... resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last...

  12. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pohl Martina

    2010-10-01

    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  13. Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

    International Nuclear Information System (INIS)

    Schallreuter, Karin U.; Wazir, Umar; Kothari, Sonal; Gibbons, Nicholas C.J.; Moore, Jeremy; Wood, John M.

    2004-01-01

    Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H 2 O 2 , we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1 x 10 -3 M), the V max for enzyme activity increased 4-fold by H 2 O 2 (>2.0 x 10 -3 M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp 120 residue to 5-OH-Trp 120 by H 2 O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2 O 2 in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H 2 O 2 by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis

  14. Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry.

    Science.gov (United States)

    Song, Hongjian; Olsen, Ole H; Persson, Egon; Rand, Kasper D

    2014-12-19

    Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Detection of bleeding disorders in Lebanon: outcomes of a pilot programme.

    Science.gov (United States)

    Djambas Khayat, C; Samaha, H; Noun, P; Bakhos Asmar, J D; Taher, A; Adib, S; Inati, A; Sakr, S

    2014-03-01

    To promote management and awareness of bleeding disorders in Lebanon, a pilot programme was launched in 2009 by the Lebanese Hemophilia Association assisted by World Federation of Hemophilia. The aim of this study was to diagnose patients with bleeding disorders and to assess the potential challenges in implementing a screening programme. The pilot project was launched in 26 social health centres in the Bekaa valley. The study tools included the evaluation of the Tossetto Bleeding Score and the Pictorial Bleeding Assessment Chart (PBAC) for menstruation. Persons with a bleeding score higher than 2 and PBAC higher than 185 were eligible for further blood tests including the prothrombin time, partial thromboplastin time, complete blood count, bleeding time and von Willebrand ristocetin cofactor activity. 643 patients were enrolled, of whom 60.6% were women. Overall, 91 persons had an abnormal score. 50 eligible patients were tested: 32 had normal tests, nine new patients with severe Von Willebrand were discovered, 4 had VW:RiCo of 40, 3 prolonged APTT and 2 thrombocytopaenia. There was a clear correlation between the severity of the score and the willingness to perform the tests (P = 0.02). Women were reluctant to participate fully when investigators were men. The probability of adherence to the screening protocol is significantly increased when directed by women health care professional. For patients with milder forms, global screening programmes were neither feasible nor acceptable but those more severely affected have to be identified. Providers are crucial in preselecting patients with blood problems who are not coping well. © 2013 John Wiley & Sons Ltd.

  16. Is anti-platelet therapy needed in continuous flow left ventricular assist device patients? A single-centre experience.

    Science.gov (United States)

    Litzler, Pierre-Yves; Smail, Hassiba; Barbay, Virginie; Nafeh-Bizet, Catherine; Bouchart, François; Baste, Jean-Marc; Abriou, Caroline; Bessou, Jean-Paul

    2014-01-01

    We report our 5-year experience of continuous flow left ventricular assist device (LVAD) implantation without the use of anti-platelet therapy. Between February 2006 and September 2011, 27 patients (26 men; 1 woman) were implanted with a continuous flow LVAD (HeartMate II, Thoratec Corporation, Pleasanton, CA, USA). The mean age was 55.7 ± 9.9 years. The mean duration of support was 479 ± 436 (1-1555) days with 35.4 patient-years on support. Twenty-one patients were implanted as a bridge to transplantation and 6 for destination therapy. The anticoagulation regimen was fluindione for all patients, with aspirin for only 4 patients. At the beginning of our experience, aspirin was administered to 4 patients for 6, 15, 60 and 460 days. Due to gastrointestinal (GI) bleeding and epistaxis, aspirin was discontinued, and since August 2006, no patients have received anti-platelet therapy. At 3 years, the survival rate during support was 76%. The most common postoperative adverse event was GI bleeding (19%) and epistaxis (30%) (median time: 26 days) for patients receiving fluindione and aspirin. The mean International Normalized Ratio (INR) was 2.58 ± 0.74 during support. Fifteen patients have been tested for acquired Von Willebrand disease. A diminished ratio of collagen-binding capacity and ristocetin cofactor activity to Von Willebrand factor antigen was observed in 7 patients. In the postoperative period, 2 patients presented with ischaemic stroke at 1 and 8 months. One of these 2 patients had a previous history of carotid stenosis with ischaemic stroke. There were no patients with haemorrhagic stroke, transient ischaemic attack or pump thrombosis. The event rate of stroke (ischaemic and haemorrhagic) per patient-year was 0.059 among the patients without aspirin with fluindione regimen only. A fluindione regimen without aspirin in long-duration LVAD support appears to not increase thromboembolic events and could lead to a diminished risk of haemorrhagic stroke.

  17. Comparative analysis of von Willebrand factor profiles after implantation of left ventricular assist device and total artificial heart.

    Science.gov (United States)

    Reich, H J; Morgan, J; Arabia, F; Czer, L; Moriguchi, J; Ramzy, D; Esmailian, F; Lam, L; Dunhill, J; Volod, O

    2017-08-01

    Essentials Bleeding is a major source of morbidity during mechanical circulatory support. von Willebrand factor (VWF) multimer loss may contribute to bleeding. Different patterns of VWF multimer loss were seen with the two device types. This is the first report of total artificial heart associated VWF multimer loss. Background Bleeding remains a challenge during mechanical circulatory support and underlying mechanisms are incompletely understood. Functional von Willebrand factor (VWF) impairment because of loss of high-molecular-weight multimers (MWMs) produces acquired von Willebrand disease (VWD) after left ventricular assist device (LVAD). Little is known about VWF multimers with total artificial hearts (TAHs). Here, VWF profiles with LVADs and TAHs are compared using a VWD panel. Methods VWD evaluations for patients with LVAD or TAH (2013-14) were retrospectively analyzed and included: VWF activity (ristocetin cofactor, VWF:RCo), VWF antigen (VWF:Ag), ratio of VWF:RCo to VWF:Ag, and quantitative VWF multimeric analysis. Results Twelve patients with LVADs and 12 with TAHs underwent VWD evaluation. All had either normal (47.8%) or elevated (52.2%) VWF:RCo, normal (26.1%) or elevated (73.9%) VWF:Ag and 50.0% were disproportional (ratio ≤ 0.7). Multimeric analysis showed abnormal patterns in all patients with LVADs: seven with high MWM loss; five with highest MWM loss. With TAH, 10/12 patients had abnormal patterns: all with highest MWM loss. High MWM loss correlated with presence of LVAD and highest MWM loss with TAH. Increased low MWMs were detected in 22/24. Conclusion Using VWF multimeric analysis, abnormalities after LVAD or TAH were detected that would be missed with measurements of VWF level alone: loss of high MWM predominantly in LVAD, loss of highest MWM in TAH, and elevated levels of low MWM in both. This is the first study to describe TAH-associated highest MWM loss, which may contribute to bleeding. © 2017 International Society on Thrombosis and

  18. Clinical and laboratory variability in a cohort of patients diagnosed with type 1 VWD in the United States.

    Science.gov (United States)

    Flood, Veronica H; Christopherson, Pamela A; Gill, Joan Cox; Friedman, Kenneth D; Haberichter, Sandra L; Bellissimo, Daniel B; Udani, Rupa A; Dasgupta, Mahua; Hoffmann, Raymond G; Ragni, Margaret V; Shapiro, Amy D; Lusher, Jeanne M; Lentz, Steven R; Abshire, Thomas C; Leissinger, Cindy; Hoots, W Keith; Manco-Johnson, Marilyn J; Gruppo, Ralph A; Boggio, Lisa N; Montgomery, Kate T; Goodeve, Anne C; James, Paula D; Lillicrap, David; Peake, Ian R; Montgomery, Robert R

    2016-05-19

    von Willebrand disease (VWD) is the most common inherited bleeding disorder, and type 1 VWD is the most common VWD variant. Despite its frequency, diagnosis of type 1 VWD remains the subject of debate. In order to study the spectrum of type 1 VWD in the United States, the Zimmerman Program enrolled 482 subjects with a previous diagnosis of type 1 VWD without stringent laboratory diagnostic criteria. von Willebrand factor (VWF) laboratory testing and full-length VWF gene sequencing was performed for all index cases and healthy control subjects in a central laboratory. Bleeding phenotype was characterized using the International Society on Thrombosis and Haemostasis bleeding assessment tool. At study entry, 64% of subjects had VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity below the lower limit of normal, whereas 36% had normal VWF levels. VWF sequence variations were most frequent in subjects with VWF:Ag <30 IU/dL (82%), whereas subjects with type 1 VWD and VWF:Ag ≥30 IU/dL had an intermediate frequency of variants (44%). Subjects whose VWF testing was normal at study entry had a similar rate of sequence variations as the healthy controls (14%). All subjects with severe type 1 VWD and VWF:Ag ≤5 IU/dL had an abnormal bleeding score (BS), but otherwise BS did not correlate with VWF:Ag. Subjects with a historical diagnosis of type 1 VWD had similar rates of abnormal BS compared with subjects with low VWF levels at study entry. Type 1 VWD in the United States is highly variable, and bleeding symptoms are frequent in this population. © 2016 by The American Society of Hematology.

  19. Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weber, Heike E; Gottardi, Manuela; Brückner, Christine; Oreb, Mislav; Boles, Eckhard; Tripp, Joanna

    2017-05-15

    Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate

  20. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    Science.gov (United States)

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  1. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Durand, Fabien [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Stines-Chaumeil, Claire [Universite de Bordeaux, CNRS, Institut de Biochimie et de Genetique Cellulaires, 1 rue Camille Saint Saens, 33077 Bordeaux Cedex (France); Flexer, Victoria [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Andre, Isabelle [Universite de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France); CNRS, UMR5504, F-31400 Toulouse (France); INRA, UMR 792 Ingenierie des Systemes Biologiques et des Procedes, F-31400 Toulouse (France); Mano, Nicolas, E-mail: mano@crpp-bordeaux.cnrs.fr [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France)

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  2. Catalytic Water Oxidation by a Bio-inspired Nickel Complex with Redox Active Ligand

    Science.gov (United States)

    Wang, Dong; Bruner, Charlie O.

    2017-01-01

    The oxidation of water to dioxygen is important in natural photosynthesis. One of nature’s strategies for managing such multi-electron transfer reactions is to employ redox active metal-organic cofactor arrays. One prototype example is the copper-tyrosinate active site found in galactose oxidase. In this work, we have implemented such a strategy to develop a bio-inspired nickel-phenolate complex capable of catalyzing the oxidation of water to O2 electrochemically at neutral pH with a modest overpotential. The employment of the redox-active ligand turned out to be a useful strategy to avoid the formation of high-valent nickel intermediates while a reasonable turnover rate (0.15 s−1) is retained. PMID:29099176

  3. Catalytic Water Oxidation by a Bio-inspired Nickel Complex with a Redox-Active Ligand.

    Science.gov (United States)

    Wang, Dong; Bruner, Charlie O

    2017-11-20

    The oxidation of water (H 2 O) to dioxygen (O 2 ) is important in natural photosynthesis. One of nature's strategies for managing such multi-electron transfer reactions is to employ redox-active metal-organic cofactor arrays. One prototype example is the copper tyrosinate active site found in galactose oxidase. In this work, we have implemented such a strategy to develop a bio-inspired nickel phenolate complex capable of catalyzing the oxidation of H 2 O to O 2 electrochemically at neutral pH with a modest overpotential. Employment of the redox-active ligand turned out to be a useful strategy to avoid the formation of high-valent nickel intermediates while a reasonable turnover rate (0.15 s -1 ) is retained.

  4. Molecular Basis for the Regulation of the H3K4 Methyltransferase Activity of PRDM9

    Directory of Open Access Journals (Sweden)

    Hong Wu

    2013-10-01

    Full Text Available PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2 and S-adenosylhomocysteine (AdoHcy, which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4 and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

  5. The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

    2018-01-26

    The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

  6. Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis.

    Science.gov (United States)

    Luthra, Pratibha Mehta; Singh, Satendra

    2010-05-01

    Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.

  7. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

    Science.gov (United States)

    Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

    2012-01-29

    Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

  8. Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication.

    Science.gov (United States)

    Merli, Marcelo L; Cirulli, Brenda A; Menéndez-Bravo, Simón M; Cricco, Julia A

    2017-06-27

    Trypanosoma cruzi , the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients' availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite's mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase-type aa 3 (C c O) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic C c O, is synthesized via two sequential enzymatic reactions catalyzed by heme O synthase (HOS) and heme A synthase (HAS). Previously, TcCox10 and TcCox15 ( Trypanosoma cruzi Cox10 and Cox15 proteins) were identified in T. cruzi They presented HOS and HAS activity, respectively, when they were expressed in yeast. Here, we present the first characterization of TcCox15 in T. cruzi , confirming its role as HAS. It was differentially detected in the different T. cruzi stages, being more abundant in the replicative forms. This regulation could reflect the necessity of more heme A synthesis, and therefore more C c O activity at the replicative stages. Overexpression of a non-functional mutant caused a reduction in heme A content. Moreover, our results clearly showed that this hindrance in the heme A synthesis provoked a reduction on C c O activity and, in consequence, an impairment on T. cruzi survival, proliferation and infectivity. This evidence supports that T. cruzi depends on the respiratory chain activity along its life cycle, being C c O an essential terminal oxidase. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. Bora and Aurora-A continue to activate Plk1 in mitosis.

    Science.gov (United States)

    Bruinsma, Wytse; Macurek, Libor; Freire, Raimundo; Lindqvist, Arne; Medema, René H

    2014-02-15

    Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora-Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora-Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.

  10. COBRA1 inhibits AP-1 transcriptional activity in transfected cells

    International Nuclear Information System (INIS)

    Zhong Hongjun; Zhu Jianhua; Zhang Hao; Ding Lihua; Sun Yan; Huang Cuifen; Ye Qinong

    2004-01-01

    Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis

  11. Naturally occurring cobalamins have antimalarial activity.

    Science.gov (United States)

    Chemaly, Susan M; Chen, Chien-Teng; van Zyl, Robyn L

    2007-05-01

    The acquisition of resistance by malaria parasites towards existing antimalarials has necessitated the development of new chemotherapeutic agents. The effect of vitamin B(12) derivatives on the formation of beta-haematin (synthetic haemozoin) was determined under conditions similar to those in the parasitic food vacuole (using chloroquine, a known inhibitor of haemozoin formation for comparison). Adenosylcobalamin (Ado-cbl), methylcobalamin (CH(3)-cbl) and aquocobalamin (H(2)O-cbl) were approximately forty times more effective inhibitors of beta-haematin formation than chloroquine, cyanocobalamin (CN-cbl) was slightly more inhibitory than chloroquine, while dicyanocobinamide had no effect. It is proposed that the cobalamins exert their inhibitory effect on beta-haematin formation by pi-interactions of their corrin ring with the Fe(III)-protoporphyrin ring and by hydrogen-bonding using their 5,6-dimethylbenzimidazole/ribose/sugar side-chain. The antimalarial activity for the cobalamins (Ado-cbl>CH(3)-cbl>H(2)O-cbl>CN-cbl) was found to be less than that for chloroquine or quinine. Ado-cbl, CH(3)-cbl and CN-cbl do not accumulate in the parasite food vacuole by pH trapping, but H(2)O-cbl does. Unlike humans, the malaria parasite has only one enzyme that uses cobalamin as a cofactor, namely methionine synthase, which is important for growth and metabolism. Thus cobalamins in very small amounts are necessary for Plasmodium falciparum growth but in larger amounts they display antimalarial properties.

  12. The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

    Directory of Open Access Journals (Sweden)

    Anna C. Maurer

    2018-05-01

    Full Text Available Summary: The adeno-associated virus (AAV vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

  13. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  14. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2012-02-01

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  15. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2009-02-27

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  16. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    International Nuclear Information System (INIS)

    Penzkofer, A.; Tanwar, M.; Veetil, S.K.; Kateriya, S.; Stierl, M.; Hegemann, P.

    2013-01-01

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction

  17. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg (Germany); Tanwar, M.; Veetil, S.K.; Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Stierl, M.; Hegemann, P. [Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany)

    2013-09-23

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

  18. Acquired von Willebrand syndrome: A rare disorder of heterogeneous etiology

    Directory of Open Access Journals (Sweden)

    P Kasatkar

    2013-01-01

    Full Text Available Context: Acquired von Willebrand syndrome (AVWS is a rare bleeding disorder that mimics the inherited form of von Willebrand disease (VWD in terms of laboratory findings and clinical presentation. Aims: To study the etiology of acquired VWD. Settings and Design: The patients referred from various hospitals in and out of Mumbai were included in the study. Materials and Methods: Six patients with AVWS diagnosed at this center over the last 10 years were analyzed against 171 patients with inherited VWD. The differential diagnosis of AVWS was made based on reduced levels of von Willebrand antigen and von Willebrand ristocetin cofactor, decrease in ristocetin induced platelet aggregation, absence of correction in mixing studies with no prior history of bleeding problems and a negative family history for bleeding disorders. Results: In three patients, the disease was associated with systematic lupus erythematosus, out of which one was also associated with Kikuchi lymphadenitis and second with autoimmune hemolytic anemia. Fourth case was associated with hypothyroidism and fifth was a case of dermatitis and vitiligo. The last patient was a case of hemophilia A with Burkitts lymphoma, who developed autoantibodies to von Willebrand factor. Except two patients, all other patients responded to immune suppressive therapy with corticosteroids, while the patient with hypothyroidism responded to oral thyroxine. Conclusion: AVWS is a rare condition and may often be missed or diagnosed as inherited disease associated with heterogeneous disease conditions.

  19. Venom Protein C activators as diagnostic agents for defects of protein C System.

    Science.gov (United States)

    Ramzan, Faiqah; Asmat, Andleeb

    2018-06-18

    Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Matrix mechanics controls FHL2 movement to the nucleus to activate p21 expression

    Science.gov (United States)

    Nakazawa, Naotaka; Sathe, Aneesh R.; Shivashankar, G. V.; Sheetz, Michael P.

    2016-01-01

    Substrate rigidity affects many physiological processes through mechanochemical signals from focal adhesion (FA) complexes that subsequently modulate gene expression. We find that shuttling of the LIM domain (domain discovered in the proteins, Lin11, Isl-1, and Mec-3) protein four-and-a-half LIM domains 2 (FHL2) between FAs and the nucleus depends on matrix mechanics. In particular, on soft surfaces or after the loss of force, FHL2 moves from FAs into the nucleus and concentrates at RNA polymerase (Pol) II sites, where it acts as a transcriptional cofactor, causing an increase in p21 gene expression that will inhibit growth on soft surfaces. At the molecular level, shuttling requires a specific tyrosine in FHL2, as well as phosphorylation by active FA kinase (FAK). Thus, we suggest that FHL2 phosphorylation by FAK is a critical, mechanically dependent step in signaling from soft matrices to the nucleus to inhibit cell proliferation by increasing p21 expression. PMID:27742790

  1. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

    Science.gov (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

    2002-11-01

    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  2. In vivo effects of radioactive properties of Tl-201 on human carbonic anhydrase activity

    Science.gov (United States)

    Sahin, Ali; Senturk, Murat

    2017-04-01

    Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3- and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. The most useful radioisotope, Tl-201, decays by electron capture, emitting Hg X-rays ( 70-80 keV), and photons of 135 and 167 keV in 10% total abundance. Therefore, it has good imaging characteristics without excessive patient radiation dose. It is the most popular isotope used for thallium 201 nuclear cardiac stress tests. In the present study, In vivo inhibitory effect of Tl-201 (Thallium-201) on human erythrocyte carbonic anhydrase (CA) activity were investigated.

  3. Engineering cofactor and ligand binding in an artificial neuroglobin

    Science.gov (United States)

    Zhang, Lei

    HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.

  4. System wide cofactor turnovers can propagate metabolic stability between pathways

    DEFF Research Database (Denmark)

    Yang, Y.; Guan, Y.H.; Villadsen, John

    2016-01-01

    . Furthermore, we elaborated the criteria to tell if a multi-enzyme over-all reaction path is of in vivo nature or not at the metabolic level. As new findings, we discovered that there are interactions between the enzyme feedback inhibition and the CI turnover, and such interactions may well lead to metabolic...

  5. Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

    NARCIS (Netherlands)

    Machielsen, M.P.; Looger, L.L.; Raedts, J.G.J.; Dijkhuizen, S.; Hummel, W.; Henneman, H.G.; Daussmann, T.; Oost, van der J.

    2009-01-01

    The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for

  6. Contaminants as viral cofactors: assessing indirect population effects

    Science.gov (United States)

    Springman, Katherine R.; Kurath, Gael; Anderson, James J.; Emlen, John M.

    2005-01-01

    Current toxicological methods often miss contaminant effects, particularly when immune suppression is involved. The failure to recognize and evaluate indirect and sublethal effects severely limits the applicability of those methods at the population level. In this study, the Vitality model is used to evaluate the population level effects of a contaminant exerting only indirect, sublethal effects at the individual level. Juvenile rainbow trout (Oncorhynchus mykiss) were injected with 2.5 or 10.0 mg/kg doses of the model CYP1A inducer, β-naphthoflavone (BNF) as a pre-stressor, then exposed to a challenge dose of 102 or 104 pfu/fish of infectious hematopoietic necrosis virus (IHNV), an important viral pathogen of salmonids in North America. At the end of the 28-d challenge, the mortality data were processed according to the Vitality model which indicated that the correlation between the average rate of vitality loss and the pre-stressor dose was strong:R2 = 0.9944. Average time to death and cumulative mortality were dependent on the BNF dose, while no significant difference between the two viral dosages was shown, implying that the history of the organism at the time of stressor exposure is an important factor in determining the virulence or toxicity of the stressor. The conceptual framework of this model permits a smoother transfer of results to a more complex stratum, namely the population level, which allows the immunosuppressive results generated by doses of a CYP1A inducer that more accurately represent the effects elicited by environmentally-relevant contaminant concentrations to be extrapolated to target populations. The indirect effects of other environmental contaminants with similar biotransformation pathways, such as polycyclic aromatic hydrocarbons (PAH), could be assessed and quantified with this model and the results applied to a more complex biological hierarchy.

  7. Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

    Directory of Open Access Journals (Sweden)

    Eriko Kamiki

    2018-05-01

    Full Text Available p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5, a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice.

  8. Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

    Science.gov (United States)

    Kamiki, Eriko; Boehringer, Roman; Polygalov, Denis; Ohshima, Toshio; McHugh, Thomas J.

    2018-01-01

    p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice. PMID:29867369

  9. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    DEFF Research Database (Denmark)

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively....... The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been...... be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation...

  10. AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

    Science.gov (United States)

    Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

    2018-04-01

    The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

  11. Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

    Directory of Open Access Journals (Sweden)

    Katharina Hahn

    2017-12-01

    Full Text Available l-Amino acid oxidases (l-AAO catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

  12. Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities.

    Science.gov (United States)

    Sakane, Rie; Kimura, Kimito; Hirota, Yoshihisa; Ishizawa, Michiyasu; Takagi, Yuta; Wada, Akimori; Kuwahara, Shigefumi; Makishima, Makoto; Suhara, Yoshitomo

    2017-11-01

    Vitamin K is an essential cofactor of γ-glutamylcarboxylase as related to blood coagulation and bone formation. Menaquinone-4, one of the vitamin K homologues, is biosynthesized in the body and has various biological activities such as being a ligand for steroid and xenobiotic receptors, protection of neuronal cells from oxidative stress, and so on. From this background, we focused on the role of menaquinone in the differentiation activity of progenitor cells into neuronal cells and we synthesized novel vitamin K derivatives with modification of the ω-terminal side chain. We report here new vitamin K analogues, which introduced an alkylated phenyl group at the ω-terminal side chain. These compounds exhibited potent differentiation activity as compared to control. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

    Directory of Open Access Journals (Sweden)

    Patrick E F O'Rourke

    Full Text Available 4-Amino-4-deoxychorismate lyase (PabC catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5'-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp(2 to sp(3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of

  14. In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra Processing Waste

    Directory of Open Access Journals (Sweden)

    Hafiz Ansar Rasul Suleria

    2016-12-01

    Full Text Available Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT, activated partial thromboplastin time (aPTT, and thromboelastography (TEG. All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

  15. In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste.

    Science.gov (United States)

    Suleria, Hafiz Ansar Rasul; Hines, Barney M; Addepalli, Rama; Chen, Wei; Masci, Paul; Gobe, Glenda; Osborne, Simone A

    2016-12-31

    Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone ( Haliotis rubra ) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

  16. Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

    Science.gov (United States)

    Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

    2007-08-03

    Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

  17. TALE factors poise promoters for activation by Hox proteins.

    Science.gov (United States)

    Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

    2014-01-27

    Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Thermophilic archaea activate butane via alkyl-coenzyme M formation.

    Science.gov (United States)

    Laso-Pérez, Rafael; Wegener, Gunter; Knittel, Katrin; Widdel, Friedrich; Harding, Katie J; Krukenberg, Viola; Meier, Dimitri V; Richter, Michael; Tegetmeyer, Halina E; Riedel, Dietmar; Richnow, Hans-Hermann; Adrian, Lorenz; Reemtsma, Thorsten; Lechtenfeld, Oliver J; Musat, Florin

    2016-11-17

    The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C 1 -compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C 4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase and reversible C 1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

  19. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    International Nuclear Information System (INIS)

    Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

    1986-01-01

    The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6- 3 H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6- 3 H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells

  20. Antiandrogenic activity of phthalate mixtures: Validity of concentration addition

    International Nuclear Information System (INIS)

    Christen, Verena; Crettaz, Pierre; Oberli-Schrämmli, Aurelia; Fent, Karl

    2012-01-01

    Phthalates and bisphenol A have very widespread use leading to significant exposure of humans. They are suspected to interfere with the endocrine system, including the androgen, estrogen and the thyroid hormone system. Here we analyzed the antiandrogenic activity of six binary, and one ternary mixture of phthalates exhibiting complete antiandrogenic dose–response curves, and binary mixtures of phthalates and bisphenol A at equi-effective concentrations of EC 10 , EC 25 and EC 50 in MDA-kb2 cells. Mixture activity followed the concentration addition (CA) model with a tendency to synergism at high and antagonism at low concentrations. Isoboles and the toxic unit approach (TUA) confirmed the additive to synergistic activity of the binary mixtures BBP + DBP, DBP + DEP and DEP + BPA at high concentrations. Both methods indicate a tendency to antagonism for the EC 10 mixtures BBP + DBP, BBP + DEP and DBP + DEP, and the EC 25 mixture of DBP + BPA. A ternary mixture revealed synergism at the EC 50 , and weak antagonistic activity at the EC 25 level by the TUA. A mixture of five phthalates representing a human urine composition and reflecting exposure to corresponding parent compounds showed no antiandrogenic activity. Our study demonstrates that CA is an appropriate concept to account for mixture effects of antiandrogenic phthalates and bisphenol A. The interaction indicates a departure from additivity to antagonism at low concentrations, probably due to interaction with the androgen receptor and/or cofactors. This study emphasizes that a risk assessment of phthalates should account for mixture effects by applying the CA concept. -- Highlights: ► Antiandrogenic activity of mixtures of 2 and 3 phthalates are assessed in MDA-kb2 cells. ► Mixture activities followed the concentration addition model. ► A tendency to synergism at high and antagonism at low levels occurred.

  1. Antiandrogenic activity of phthalate mixtures: Validity of concentration addition

    Energy Technology Data Exchange (ETDEWEB)

    Christen, Verena [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Crettaz, Pierre; Oberli-Schrämmli, Aurelia [Swiss Federal Office of Public Health, Division Chemical Products, 3003 Bern (Switzerland); Fent, Karl, E-mail: karl.fent@bluewin.ch [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich (ETH Zürich), Department of Environmental Sciences, 8092 Zürich (Switzerland)

    2012-03-01

    Phthalates and bisphenol A have very widespread use leading to significant exposure of humans. They are suspected to interfere with the endocrine system, including the androgen, estrogen and the thyroid hormone system. Here we analyzed the antiandrogenic activity of six binary, and one ternary mixture of phthalates exhibiting complete antiandrogenic dose–response curves, and binary mixtures of phthalates and bisphenol A at equi-effective concentrations of EC{sub 10}, EC{sub 25} and EC{sub 50} in MDA-kb2 cells. Mixture activity followed the concentration addition (CA) model with a tendency to synergism at high and antagonism at low concentrations. Isoboles and the toxic unit approach (TUA) confirmed the additive to synergistic activity of the binary mixtures BBP + DBP, DBP + DEP and DEP + BPA at high concentrations. Both methods indicate a tendency to antagonism for the EC{sub 10} mixtures BBP + DBP, BBP + DEP and DBP + DEP, and the EC{sub 25} mixture of DBP + BPA. A ternary mixture revealed synergism at the EC{sub 50}, and weak antagonistic activity at the EC{sub 25} level by the TUA. A mixture of five phthalates representing a human urine composition and reflecting exposure to corresponding parent compounds showed no antiandrogenic activity. Our study demonstrates that CA is an appropriate concept to account for mixture effects of antiandrogenic phthalates and bisphenol A. The interaction indicates a departure from additivity to antagonism at low concentrations, probably due to interaction with the androgen receptor and/or cofactors. This study emphasizes that a risk assessment of phthalates should account for mixture effects by applying the CA concept. -- Highlights: ► Antiandrogenic activity of mixtures of 2 and 3 phthalates are assessed in MDA-kb2 cells. ► Mixture activities followed the concentration addition model. ► A tendency to synergism at high and antagonism at low levels occurred.

  2. Active Teachers - Active Students

    DEFF Research Database (Denmark)

    as an initiative from the Polytechnic in Nantes, France and the University the Los Andes in Bogota, Colombia. The objective was to start a world wide collaboration allowing teachers in engineering to learn from each other about their experiences with active learning. In this thirteenth edition, ALE joins forces...... with the International Research Symposium on Problem Based Learning (IRSPB) and the International Symposium on Project Approaches in Engineering Education (PAEE) to organise the first International Joint Conference on the Learner in Engineering Education (IJCLEE 2015) hosted by Mondragon University, in San Sebastian...

  3. Is activation analysis still active?

    International Nuclear Information System (INIS)

    Chai Zhifang

    2001-01-01

    This paper reviews some aspects of neutron activation analysis (NAA), covering instrumental neutron activation analysis (INAA), k 0 method, prompt gamma-ray neutron activation analysis (PGNAA), radiochemical neutron activation analysis (RNAA) and molecular activation analysis (MAA). The comparison of neutron activation analysis with other analytical techniques are also made. (author)

  4. Fucosylated chondroitin sulfate oligosaccharides exert anticoagulant activity by targeting at intrinsic tenase complex with low FXII activation: Importance of sulfation pattern and molecular size.

    Science.gov (United States)

    Li, Junhui; Li, Shan; Yan, Lufeng; Ding, Tian; Linhardt, Robert J; Yu, Yanlei; Liu, Xinyue; Liu, Donghong; Ye, Xingqian; Chen, Shiguo

    2017-10-20

    Fucosylated chondroitin sulfates (fCSs) are structurally unusual glycosaminoglycans isolated from sea cucumbers that exhibit potent anticoagulant activity. These fCSs were isolated from sea cucumber, Isostichopus badionotus and Pearsonothuria graeffei. Fenton reaction followed by gel filtration chromatography afforded fCS oligosaccharides, with different sulfation patterns identified by mass and NMR spectroscopy, and these were used to clarify the relationship between the structures and the anticoagulant activities of fCSs. In vitro activities were measured by activated partial thromboplastin time (APTT), thrombin time (TT), thrombin and factor Xa inhibition, and activation of FXII. The results showed that free radicals preferentially acted on GlcA residues affording oligosaccharides that were purified from both fCSs. The inhibition of thrombin and factor X activities, mediated through antithrombin III and heparin cofactor II of fCSs oligosaccharides were affected by their molecular weight and fucose branches. Oligosaccharides with different sulfation patterns of the fucose branching had a similar ability to inhibit the FXa by the intrinsic factor Xase (factor IXa-VIIIa complex). Oligosaccharides with 2,4-O-sulfo fucose branches from fCS-Ib showed higher activities than ones with 3,4-O-disulfo branches obtained from fCS-Pg. Furthermore, a heptasaccharide is the minimum size oligosaccharide required for anticoagulation and FXII activation. This activity was absent for fCS oligosaccharides smaller than nonasaccharides. Molecular size and fucose branch sulfation are important for anticoagulant activity and reduction of size can reverse the activation of FXII caused by native fCSs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Increased tumour ascorbate is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in human colorectal cancer

    Directory of Open Access Journals (Sweden)

    Caroline eKuiper

    2014-02-01

    Full Text Available Ascorbate is a co-factor for the hydroxylases that regulate the transcription factor hypoxia-inducible factor (HIF-1, which provides cancer cells with a metabolic and survival advantage in the hypoxic environment of solid tumors. However, whether ascorbate affects tumor development is a highly debated issue. We aimed to determine whether tumor ascorbate was associated with HIF-1 activation and patient disease-free survival. In this study we undertook a retrospective observational analysis of tissue-banked tumor and paired normal tissue from 49 colorectal cancer patients, measuring ascorbate levels, HIF-1α and its downstream gene products BNIP3 and VEGF. Patient survival was monitored for the first six years after surgery. We found that ascorbate levels were lower in tumor tissue compared to normal tissue (p< 0.001 but overall levels varied considerably. HIF-1α, VEGF and BNIP3 were elevated in tumor samples (p< 0.01. There was an inverse relationship between tumor ascorbate content and HIF-1 pathway activation (p=0.002 and tumor size (p=0.018. Higher tumor ascorbate content was associated with significantly improved disease-free survival in the first 6 years after surgery (p=0.006, with 141 - 1,094 additional disease free days. This was independent of tumor grade and stage. Survival advantage was associated with the amount of ascorbate in the tumor, but not with the amount in adjacent normal tissue. Our results demonstrate that higher tumor ascorbate content is associated decreased HIF-1 activation, most likely due to the co-factor activity of ascorbate for the regulatory HIF hydroxylases. Our findings support the need for future studies to determine whether raising tumor ascorbate is possible with clinical intervention and whether this results in modification of hydroxylase-dependent pathways in the tumor.

  6. Dynamic regulation of Drosophila nuclear receptor activity in vivo.

    Science.gov (United States)

    Palanker, Laura; Necakov, Aleksandar S; Sampson, Heidi M; Ni, Ruoyu; Hu, Chun; Thummel, Carl S; Krause, Henry M

    2006-09-01

    Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.

  7. Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

    International Nuclear Information System (INIS)

    Shibata, Y.; Abiko, Y.; Ohno, H.; Araki, T.; Takiguchi, H.

    1988-01-01

    Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A 2 had an optimum pH at 4.5, and enzyme activation was observed in Ca ++ -free medium; but the maximum activity was found at 0.5 mM Ca ++ concentration. The Km value for PC of acidic PLase A 2 was 4.2 μM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A 2 in light of the uncompetitive manner of Ca ++ action. Furthermore, the release of [ 3 H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca ++ at pH 4.5. These data suggest that the acid PLase A 2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A 2 properties are opposite to those found for lysosomal PLase A 2

  8. Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

    Science.gov (United States)

    Ferreira, G; Weiss, W P

    2007-03-01

    Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

  9. The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90.

    Science.gov (United States)

    Bartolini, Manuela; Wainer, Irving W; Bertucci, Carlo; Andrisano, Vincenza

    2013-01-25

    Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Growth and Extracellular Carbonic Anhydrase Activity of Zooxanthellae Symbiodinium sp. in Response of Zinc Enrichment

    Directory of Open Access Journals (Sweden)

    WIDIASTUTI KARIM

    2011-12-01

    Full Text Available Coral reef communities contain a wide variety of mutualistic associations none more important than the relationship between corals and their symbiotic dinoflagellates of the genus Symbiodinium sp., commonly referred to as zooxanthellae. The function of Zinc (Zn as cofactor of several enzyme systems such as extracellular carbonic anhydrase (extracellular CA which catalyzes the interconversion of HCO3- and CO2. Concentrations of dissolved Zn in oligothropic waters are often very low therefore may limit the growth of zooxanthellae and their ability to fix CO2 from seawater via the carbonic anhydrase. The aim of this research is to investigate the effect of various concentrations of Zn on the growth and extracellular CA activity in zooxanthellae. Cell density was monitored daily by enumeration with hemocytometer-type chamber (0.1 mm. Extracellular CA was measured in homogenized intact whole cell by a pH drift assay. Results revealed that Zn status strongly influences the growth rate and extracelullar CA activity in zooxanthellae. The specific growth rate and cell density increased two-fold whilst extracelullar CA activity increased 10.5 times higher than that in control with increasing concentrations of Zn from 0 to 80 nM, but decreased when Zn was over 80 nM. Under a concentration of 80 nM was not Zn limited culture, consequently the growth rate of zooxanthellae not dependent on CO2 concentration yet offset by extracelullar CA activity.

  11. Growth and Extracellular Carbonic Anhydrase Activity of Zooxanthellae Symbiodinium sp. in Response of Zinc Enrichment

    Directory of Open Access Journals (Sweden)

    WIDIASTUTI KARIM

    2011-12-01

    Full Text Available Coral reef communities contain a wide variety of mutualistic associations none more important than the relationship between corals and their symbiotic dinoflagellates of the genus Symbiodinium sp., commonly referred to as zooxanthellae. The function of Zinc (Zn as cofactor of several enzyme systems such as extracellular carbonic anhydrase (extracellular CA which catalyzes the interconversion of HCO3− and CO2. Concentrations of dissolved Zn in oligothropic waters are often very low therefore may limit the growth of zooxanthellae and their ability to fix CO2 from seawater via the carbonic anhydrase. The aim of this research is to investigate the effect of various concentrations of Zn on the growth and extracellular CA activity in zooxanthellae. Cell density was monitored daily by enumeration with hemocytometer-type chamber (0.1 mm. Extracellular CA was measured in homogenized intact whole cell by a pH drift assay. Results revealed that Zn status strongly influences the growth rate and extracelullar CA activity in zooxanthellae. The specific growth rate and cell density increased two-fold whilst extracelullar CA activity increased 10.5 times higher than that in control with increasing concentrations of Zn from 0 to 80 nM, but decreased when Zn was over 80 nM. Under a concentration of 80 nM was not Zn limited culture, consequently the growth rate of zooxanthellae not dependent on CO2 concentration yet offset by extracelullar CA activity.

  12. Potent Chemopreventive/Antioxidant Activity Detected in Common Spices of the Apiaceae Family.

    Science.gov (United States)

    Jeyabalan, Jeyaprakash; Aqil, Farrukh; Soper, Lisa; Schultz, David J; Gupta, Ramesh C

    2015-01-01

    Spices are used worldwide, particularly in the Asian and Middle Eastern countries, and considered protective against degenerative diseases, including cancer. Here, we report the efficacy of aqueous and non-aqueous extracts of 11 Apiaceae spices for free radical-scavenging activity and to inhibit cytochrome P450s in two separate reactions involving: 1) 4-hydroxy-17ß-estradiol (4E2), DNA, and CuCl2 and 2) 17ß-estradiol, rat liver microsomes, cofactors, DNA and CuCl2. Oxidative DNA adducts resulting from redox cycling of 4E2 were analyzed by (32)P-postlabeling. Aqueous (5 mg/ml) and non-aqueous extracts (6 mg/ml) substantially inhibited (83-98%) formation of DNA adducts in the microsomal reaction. However, in nonmicrosomal reaction, only aqueous extracts showed the inhibitory activity (83-96%). Adduct inhibition was also observed at five-fold lower concentrations of aqueous extracts of cumin (60%) and caraway (90%), and 10-fold lower concentrations of carrot seeds (76%) and ajowan (90%). These results suggests the presence of 2 groups of phytochemicals: polar compounds that have free radical-scavenging activity and lipophilic compounds that selectively inhibit P450 activity associated with estrogen metabolism. Because most of these Apiaceae spices are used widely with no known toxicity, the phytochemicals from the Apiaceae spices used in foods may be potentially protective against estrogen-mediated breast cancer.

  13. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

    Science.gov (United States)

    Perdomo, José; Jiang, Xing-Mai; Carter, Daniel R; Khachigian, Levon M; Chong, Beng H

    2012-01-01

    Friend of GATA 2 (FOG-2), a co-factor of several GATA transcription factors (GATA-4, -5 and 6), is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955) [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE), while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP) promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  14. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

    Directory of Open Access Journals (Sweden)

    José Perdomo

    Full Text Available Friend of GATA 2 (FOG-2, a co-factor of several GATA transcription factors (GATA-4, -5 and 6, is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955 [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE, while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  15. Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy*

    Science.gov (United States)

    Matzner, Ulrich; Breiden, Bernadette; Schwarzmann, Günter; Yaghootfam, Afshin; Fluharty, Arvan L.; Hasilik, Andrej; Sandhoff, Konrad; Gieselmann, Volkmar

    2009-01-01

    Arylsulfatase A (ASA) catalyzes the intralysosomal desulfation of 3-O-sulfogalactosylceramide (sulfatide) to galactosylceramide. The reaction requires saposin B (Sap B), a non-enzymatic proteinaceous cofactor which presents sulfatide to the catalytic site of ASA. The lack of either ASA or Sap B results in a block of sulfatide degradation, progressive intralysosomal accumulation of sulfatide, and the fatal lysosomal storage disease metachromatic leukodystrophy. We studied the coupled Sap B-ASA reaction in vitro using detergent-free micellar and liposomal assay systems and in vivo using cell culture models of metachromatic leukodystrophy. Under in vitro conditions, the reaction had a narrow pH optimum around pH 4.3 and was inhibited by mono- and divalent cations, phosphate and sulfite. Bis(monoacylglycero) phosphate and phosphatidic acid were activators of the reaction, underscoring a significant role of acidic phosphoglycerolipids in sphingolipid degradation. Desulfation was negligible when Sap B was substituted by Sap A, C, or D. Up to a molar ratio between Sap B and sulfatide of 1:5, an elevation of Sap B concentrations caused a sharp increase of sulfatide hydrolysis, indicating the requirement of unexpected high Sap B levels for maximum turnover. Feeding of ASA-deficient, sulfatide-storing primary mouse kidney cells with ASA caused partial clearance of sulfatide. Co-feeding of Sap B or its precursor prosaposin resulted in the lysosomal uptake of the cofactor but did not promote ASA-catalyzed sulfatide hydrolysis. This suggests that Sap B is not a limiting factor of the coupled Sap B-ASA reaction in mouse kidney cells even if sulfatide has accumulated to unphysiologically high levels. PMID:19224915

  16. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    Science.gov (United States)

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  17. Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

    Science.gov (United States)

    Latif, S A; Sheff, M F; Ribeiro, C E; Morris, D J

    1997-02-01

    We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

  18. Activated Charcoal

    Science.gov (United States)

    Common charcoal is made from peat, coal, wood, coconut shell, or petroleum. “Activated charcoal” is similar to common charcoal, but is made especially for use as a medicine. To make activated charcoal, manufacturers heat common ...

  19. THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

    Science.gov (United States)

    Sono, Takashi; Akiyama, Haruhiko; Miura, Shigenori; Deng, Jian Min; Shukunami, Chisa; Hiraki, Yuji; Tsushima, Yu; Azuma, Yoshiaki; Behringer, Richard R; Matsuda, Shuichi

    2018-07-01

    Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

  20. Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day Adventists, control subjects, and bowel cancer patients.

    Science.gov (United States)

    Macdonald, I A; Webb, G R; Mahony, D E

    1978-10-01

    Cell-free extracts were prepared from mixed fecal anaerobic bacteria grown from stools of 14 vegetarian Seventh-Day Adventists, 16 omnivorous control subjects, and eight patients recently diagnosed with cancer of the large bowel. Preparations were assayed for NAD- and NADP-dependent 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenases with bile salts and androsterone as substrates (eight substrate-cofactor combinations were tested). A significant intergroup difference was observed in the amounts of NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenase produced: bowel cancer patients exceeded controls, and controls exceeded Seventh-Day Adventists. Other enzyme activity comparisons were not significant. The pH values of the stools were significantly higher in cancer patients compared to Seventh-Day Adventists; values were 7.03 +/- 0.60 and 6.46 +/- 0.58 respectively. The pH value for controls was 6.66 +/- 0.62. A plot of pH value versus NADP-dependent 7alpha-hydroxysteroid dehydrogenase tended to separate the cancer patients from the other groups. Comparative data suggest that much of the 3alpha-hydroxysteroid dehydrogenase active against bile salt is also active against androsterone.

  1. Negative Correlation between the Diffusion Coefficient and Transcriptional Activity of the Glucocorticoid Receptor.

    Science.gov (United States)

    Mikuni, Shintaro; Yamamoto, Johtaro; Horio, Takashi; Kinjo, Masataka

    2017-08-25

    The glucocorticoid receptor (GR) is a transcription factor, which interacts with DNA and other cofactors to regulate gene transcription. Binding to other partners in the cell nucleus alters the diffusion properties of GR. Raster image correlation spectroscopy (RICS) was applied to quantitatively characterize the diffusion properties of EGFP labeled human GR (EGFP-hGR) and its mutants in the cell nucleus. RICS is an image correlation technique that evaluates the spatial distribution of the diffusion coefficient as a diffusion map. Interestingly, we observed that the averaged diffusion coefficient of EGFP-hGR strongly and negatively correlated with its transcriptional activities in comparison to that of EGFP-hGR wild type and mutants with various transcriptional activities. This result suggests that the decreasing of the diffusion coefficient of hGR was reflected in the high-affinity binding to DNA. Moreover, the hyper-phosphorylation of hGR can enhance the transcriptional activity by reduction of the interaction between the hGR and the nuclear corepressors.

  2. Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

    Science.gov (United States)

    Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

    2012-01-01

    Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

  3. Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

    Science.gov (United States)

    Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

    1999-03-01

    The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

  4. Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

    Science.gov (United States)

    Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

    2017-04-01

    Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

  5. Multiple Taf subunits of TFIID interact with Ino2 activation domains and contribute to expression of genes required for yeast phospholipid biosynthesis.

    Science.gov (United States)

    Hintze, Stefan; Engelhardt, Maike; van Diepen, Laura; Witt, Eric; Schüller, Hans-Joachim

    2017-12-01

    Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2. © 2017 John Wiley & Sons Ltd.

  6. Patterns of receptive and creative cultural activities and their association with perceived health, anxiety, depression and satisfaction with life among adults: the HUNT study, Norway.

    Science.gov (United States)

    Cuypers, Koenraad; Krokstad, Steinar; Holmen, Turid Lingaas; Skjei Knudtsen, Margunn; Bygren, Lars Olov; Holmen, Jostein

    2012-08-01

    Cultural participation has been used both in governmental health policies and as medical therapy, based on the assumption that cultural activities will improve health. Previous population studies and a human intervention study have shown that religious, social and cultural activities predict increased survival rate. The aim of this study was to analyse the association between cultural activity and perceived health, anxiety, depression and satisfaction with life in both genders. The study is based on the third population-based Nord-Trøndelag Health Study (2006-2008), including 50,797 adult participants from Nord-Trøndelag County, Norway. Data on cultural activities, both receptive and creative, perceived health, anxiety, depression and satisfaction with life were collected by comprehensive questionnaires. The logistic regression models, adjusted for relevant cofactors, show that participation in receptive and creative cultural activities was significantly associated with good health, good satisfaction with life, low anxiety and depression scores in both genders. Especially in men, attending receptive, rather than creative, cultural activities was more strongly associated with all health-related outcomes. Statistically significant associations between several single receptive, creative cultural activities and the health-related outcome variables were revealed. This population-based study suggests gender-dependent associations between cultural participation and perceived health, anxiety, depression and satisfaction with life. The results support hypotheses on the effect of cultural activities in health promotion and healthcare, but further longitudinal and experimental studies are warranted to establish a reliable cause-effect relationship.

  7. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Energy Technology Data Exchange (ETDEWEB)

    Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  8. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    International Nuclear Information System (INIS)

    Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

    2011-01-01

    Highlights: → Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. → Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. → Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. → Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex

  9. Active ageing

    Directory of Open Access Journals (Sweden)

    Frode F. Jacobsen

    2017-09-01

    Full Text Available Background: The concept of active ageing has been gaining prominence in the Nordic countries and beyond. This has been reflected in policy papers in Norway and other Nordic nations. Aims: The aim of this article is to analyse the topic of active ageing in five Norwegian White Papers (2002 to 2015 and discuss those policy documents in context of relevant research literature. Methods: A qualitative document analyses is employed focusing on how active ageing, and ageing in general, is described and which concepts are employed. No ethical approval was needed. Findings: The general theme of ageing and the specific theme of active ageing are increasingly prominent in the Norwegian White Papers studied. In all documents, some assumptions regarding ageing and active ageing seem implicit, such as independence being more important than (interdependence. ‘Productive’ activities like participation in working life are stressed, while others, like reading, watching TV or watching children playing in the street, are ignored. Conclusions: The policy documents demonstrate that the topic of active ageing is growing in importance. The documents increasingly seem to stress ‘productive’ activities – those related to working life, voluntary work or sports and physical training. They exclude activities that are meaningful for many older people, like watching their grandchildren play or reading books. Implications for practice: Practitioners in older people’s care could consider reflecting on: Government documents dealing with their own practice The prevalent concept of active ageing The trend of active ageing as a facilitating or hindering factor for good care work How present discourse on active ageing may influence their attitude towards frail older persons How they wish to relate to active ageing in their own practice

  10. Physical Activity

    DEFF Research Database (Denmark)

    Andersen, Lars Bo; Anderssen, Sigmund Alfred; Wisløff, Ulrik

    2014-01-01

    Andersen LB, Anderssen SA, Wisløff U, Hellénius M-L, Fogelholm M, Ekelund U. (Expert Group) Nordic Nutrition Recommendations 2012. Integrating nutrition and physical activity. Chapter: Physical Activity p. 195-217.Nordic Counsil of Ministers.......Andersen LB, Anderssen SA, Wisløff U, Hellénius M-L, Fogelholm M, Ekelund U. (Expert Group) Nordic Nutrition Recommendations 2012. Integrating nutrition and physical activity. Chapter: Physical Activity p. 195-217.Nordic Counsil of Ministers....

  11. Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States); Serrano, Hector; Whitman, Christian P. [Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin (United States); Mesecar, Andrew D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States)

    2008-12-01

    The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed. Trans-3-chloroacrylic acid dehalogenase (CaaD) is a critical enzyme in the trans-1, 3-dichloropropene (DCP) degradation pathway in Pseudomonas pavonaceae 170. This enzyme allows bacteria to use trans-DCP, a common component in commercially produced fumigants, as a carbon source. CaaD specifically catalyzes the fourth step of the pathway by cofactor-independent dehalogenation of a vinyl carbon–halogen bond. Previous studies have reported an X-ray structure of CaaD under acidic conditions with a covalent modification of the catalytic βPro1 residue. Here, the 1.7 Å resolution X-ray structure of CaaD under neutral (pH 6.5) conditions is reported without the presence of the covalent adduct. In this new structure, a substrate-like acetate molecule is bound within the active site in a position analogous to the putative substrate-binding site. Additionally, a catalytically important water molecule was identified, consistent with previously proposed reaction schemes. Finally, flexibility of the catalytically relevant side chain αGlu52 is observed in the structure, supporting its role in the catalytic mechanism.

  12. The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.

    Science.gov (United States)

    Cepeda, Marisa R; McGarry, Lauren; Pennington, Joseph M; Krzystek, J; Elizabeth Stroupe, M

    2018-05-28

    The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO 3 2- to S 2- . Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe 4 S 4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe 4 S 4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site. Copyright © 2018. Published by Elsevier B.V.

  13. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    Science.gov (United States)

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

  14. TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation.

    Science.gov (United States)

    Tong, Wing-Hang; Maio, Nunziata; Zhang, De-Liang; Palmieri, Erika M; Ollivierre, Hayden; Ghosh, Manik C; McVicar, Daniel W; Rouault, Tracey A

    2018-05-22

    Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)-activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)-mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders.

  15. Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds.

    Science.gov (United States)

    Rahmanpour, Rahman; Rea, Dean; Jamshidi, Shirin; Fülöp, Vilmos; Bugg, Timothy D H

    2016-03-15

    A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a β-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

    Science.gov (United States)

    Mancini, Stefano; Imlay, James A

    2015-05-01

    Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology. © 2015 John Wiley & Sons Ltd.

  17. Stearoyl-Acyl Carrier Protein and Unusual Acyl-Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin1

    Science.gov (United States)

    Schultz, David J.; Suh, Mi Chung; Ohlrogge, John B.

    2000-01-01

    Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Δ9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [14C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium × hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Δ9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction. PMID:11027717

  18. Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

    Science.gov (United States)

    Schultz, D J; Suh, M C; Ohlrogge, J B

    2000-10-01

    Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

  19. Disruption of gene pqqA or pqqB reduces plant growth promotion activity and biocontrol of crown gall disease by Rahnella aquatilis HX2.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Rahnella aquatilis strain HX2 has the ability to promote maize growth and suppress sunflower crown gall disease caused by Agrobacterium vitis, A. tumefaciens, and A. rhizogenes. Pyrroloquinoline quinone (PQQ, a cofactor of aldose and alcohol dehydrogenases, is required for the synthesis of an antibacterial substance, gluconic acid, by HX2. Mutants of HX2 unable to produce PQQ were obtained by in-frame deletion of either the pqqA or pqqB gene. In this study, we report the independent functions of pqqA and pqqB genes in relation to PQQ synthesis. Interestingly, both the pqqA and pqqB mutants of R. aquatilis eliminated the ability of strain HX2 to produce antibacterial substance, which in turn, reduced the effectiveness of the strain for biological control of sunflower crown gall disease. The mutation also resulted in decreased mineral phosphate solubilization by HX2, which reduced the efficacy of this strain as a biological fertilizer. These functions were restored by complementation with the wild-type pqq gene cluster. Additionally, the phenotypes of HX2 derivatives, including colony morphology, growth dynamic, and pH change of culture medium were impacted to different extents. Our findings suggested that pqqA and pqqB genes individually play important functions in PQQ biosynthesis and are required for antibacterial activity and phosphorous solubilization. These traits are essential for R. aquatilis efficacy as a biological control and plant growth promoting strain. This study enhances our fundamental understanding of the biosynthesis of an environmentally significant cofactor produced by a promising biocontrol and biological fertilizer strain.

  20. Activity report

    International Nuclear Information System (INIS)

    1990-11-01

    The Department of Physics and Measurement Technology, Biology and Chemistry (IFM) presents every year a progress report containing a brief description of activities in research and education within the department. The report is intended as an information for colleagues and institutions. The present report contains activities for the academic year July 1989 to June 1990

  1. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    International Nuclear Information System (INIS)

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells

  2. Seasonal variation in tissue estrogen-2/4-hydroxylases (EH) and in vitro effects of steroids on ovarian EH activity in the catfish Heteropneustes fossilis.

    Science.gov (United States)

    Chourasia, T K; Joy, K P

    2010-12-12

    A radiometric assay was used to measure microsomal EH activity from tritiated H(2)O formed during the conversion of [2,4 (3)H] estradiol-17β into catecholestrogens in the microsomal fractions of liver, brain and ovary of the catfish Heteropneustes fossilis. The validation data show that enzyme activity increased with incubation time, and substrate and cofactor (NADPH) concentrations, elicited temperature optima of 30-37°C and pH optima of 6.8-7.8. EH activity was strongly NADPH-dependent and in its absence only 13.48% activity was recorded. Liver recorded the highest enzyme activity, followed by brain and ovary. EH activity showed a significant seasonal variation with the peak activity in spawning phase and the lowest activity in resting phase. In the ovary, the follicular layer (theca and granulosa) elicited the highest activity over that of the denuded oocytes. Modulatory effects of steroids on ovarian enzyme activity were further demonstrated. The incubation of postvitellogenic follicles with 1, 10 or 100 nM concentrations of various steroids for 24 h produced varied effects on EH activity. Progesterone and 2-hydroxyestradiol-17β elicited strong suppressive effects on enzyme activity. Estrogens (E(1), E(2) and E(3)) suppressed the activity in a concentration-dependent manner. Among the progestins tested, 17,20α-dihydroxy-4-pregnen-3-one, the isomer of 17,20β-dihydroxy-4-pregnen-3-one (a teleost maturation-inducing steroid) showed the lowest depressing effect. Among androgens, the testosterone metabolite 11-ketotestosterone (functional teleost androgen) showed a high suppressing effect. Corticosteroids elicited low activity with cortisol suppressed the activity at higher concentrations. The study will form a basis to understand the physiological role of catecholestrogens in ovarian functions. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients.

    Directory of Open Access Journals (Sweden)

    Petronela Ancuta

    2008-06-01

    Full Text Available Elevated plasma lipopolysaccharide (LPS, an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD. To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+ T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

  4. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

    Directory of Open Access Journals (Sweden)

    Julia Penna-Coutinho

    Full Text Available The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2. The IC(50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

  5. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    International Nuclear Information System (INIS)

    Hu, Xu-Dong; Meng, Qing-Hui; Xu, Jia-Ying; Jiao, Yang; Ge, Chun-Min; Jacob, Asha; Wang, Ping; Rosen, Eliot M; Fan, Saijun

    2011-01-01

    Research highlights: → BTG2 associates with AR, androgen causes an increase of the interaction. → BTG2 as a co-repressor inhibits the AR-mediated transcription activity. → BTG2 inhibits the transcription activity and expression of PSA. → An intact 92 LxxLL 96 motif is essential and necessary for these activities of BTG2, while the 20 LxxLL 24 motif is not required. → Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ( 20 LxxLL 24 and 92 LxxLL 96 ), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant 20 LxxLL 24 motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant 92 LxxLL 96 motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact 92 LxxLL 96 motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  6. The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

    Science.gov (United States)

    Huber, Roland G; Fan, Hao; Bond, Peter J

    2015-10-01

    ZAP-70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD), and binding of its regulatory tandem Src homology 2 (SH2) domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

  7. The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

    Directory of Open Access Journals (Sweden)

    Roland G Huber

    2015-10-01

    Full Text Available ZAP-70 (Zeta-chain-associated protein kinase 70 is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD, and binding of its regulatory tandem Src homology 2 (SH2 domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

  8. [Active euthanasia].

    Science.gov (United States)

    Folker, A P; Hvidt, N

    1995-02-20

    The growing interest in the subject of active euthanasia in connection with the debate regarding legalization of such practices in Denmark necessitates taking a definite standpoint. The difference in concept between active and passive euthanasia is stressed, and the Dutch guidelines are reviewed. The article discusses how far the patient's autonomy should go, as it regards the consideration of self-determination as being too narrow a criterion in itself. The discussion on the quality of life is included, and the consequences of the process of expulsion as a sociological concept are considered--the risk of a patient feeling guilty for being alive and therefore feeling compelled to request active euthanasia. The changed function of the physician is underlined, and it is discussed whether active euthansia will cause a breach of confidence between the physician and his patient. In connection with the debate the following tendencies in society are emphasized: lack of clarity, increasing medicalization and utilitarian priorities.

  9. Effect of high dose thiamine therapy on activity and molecular ...

    African Journals Online (AJOL)

    Commonest form of diabetes mellitus is Type 2, treated with oral hypoglycemic agents, which often carry potential adverse effects and do not address the intracellular metabolism of glucose. Thiamine is an essential co-factor for vital subcellular enzymes and has potential to benefit Type 2 diabetics. This study was therefore ...

  10. Dual protection in sexually active women | Kleinschmidt | South ...

    African Journals Online (AJOL)

    Objective. To determine the prevalence and co-factors associated with the practice of dual protection against sexually transmitted infections (STis) and unwanted pregnancy in a cross-sectional sample of South African women. Design. Secondary analysis of cross-sectional household survey data. Methods. Statistical ...

  11. Redox activity distinguishes solid-state electron transport from solution-based electron transfer in a natural and artificial protein: cytochrome C and hemin-doped human serum albumin.

    Science.gov (United States)

    Amdursky, Nadav; Ferber, Doron; Pecht, Israel; Sheves, Mordechai; Cahen, David

    2013-10-28

    Integrating proteins in molecular electronic devices requires control over their solid-state electronic transport behavior. Unlike "traditional" electron transfer (ET) measurements of proteins that involve liquid environments and a redox cycle, no redox cofactor is needed for solid-state electron transport (ETp) across the protein. Here we show the fundamental difference between these two approaches by macroscopic area measurements, which allow measuring ETp temperature dependence down to cryogenic temperatures, via cytochrome C (Cyt C), an ET protein with a heme (Fe-porphyrin) prosthetic group as a redox centre. We compare the ETp to electrochemical ET measurements, and do so also for the protein without the Fe (with metal-free porphyrin) and without porphyrin. As removing the porphyrin irreversibly alters the protein's conformation, we repeat these measurements with human serum albumin (HSA), 'doped' (by non-covalent binding) with a single hemin equivalent, i.e., these natural and artificial proteins share a common prosthetic group. ETp via Cyt C and HSA-hemin are very similar in terms of current magnitude and temperature dependence, which suggests similar ETp mechanisms via these two systems, thermally activated hopping (with ~0.1 eV activation energy) >190 K and tunneling by superexchange Fe(3+) + e(-)), measured by electrochemistry of HSA-hemin are only 4 times lower than those for Cyt C. However, while removing the Fe redox centre from the porphyrin ring markedly affects the ET rate, it hardly changes the ETp currents through these proteins, while removing the macrocycle (from HSA, which retains its conformation) significantly reduces ETp efficiency. These results show that solid-state ETp across proteins does not require the presence of a redox cofactor, and that while for ET the Fe ion is the main electron mediator, for ETp the porphyrin ring has this function.

  12. Omega-6 polyunsaturated fatty acids, serum zinc, delta-5- and delta-6-desaturase activities and incident metabolic syndrome.

    Science.gov (United States)

    Yary, T; Voutilainen, S; Tuomainen, T-P; Ruusunen, A; Nurmi, T; Virtanen, J K

    2017-08-01

    The associations of n-6 polyunsaturated fatty acids (PUFA) with metabolic syndrome have been poorly explored. We investigated the associations of the serum n-6 PUFA and the activities of enzymes involved in the PUFA metabolism, delta-5-desaturase (D5D) and delta-6-desaturase (D6D) with risk of incident metabolic syndrome. We also investigated whether zinc, a cofactor for these enzymes, modifies these associations. A prospective follow-up study was conducted on 661 men who were aged 42-60 years old at baseline in 1984-1989 and who were re-examined in 1998-2001. Men in the highest versus the lowest serum total omega-6 PUFA tertile had a 70% lower multivariate-adjusted risk of incident metabolic syndrome [odds ratio (OR) = 0.30; 95% confidence interval (CI) = 0.18-0.51, P trend metabolic syndrome components at the re-examinations. Most associations were attenuated after adjustment for body mass index. Finally, the associations of D6D and LA were stronger among those with a higher serum zinc concentration. Higher serum total n-6 PUFA, linoleic acid and arachidonic acid concentrations and D5D activity were associated with a lower risk of developing metabolic syndrome and higher D6D activity was associated with a higher risk. The role of zinc also needs to be investigated in other populations. © 2016 The British Dietetic Association Ltd.

  13. Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Wupeng; Stone, Everett; Zhang, Yan Jessie

    2017-02-01

    Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

  14. Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

    Directory of Open Access Journals (Sweden)

    Nor ‘Aini, A. R.

    2006-01-01

    Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

  15. Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site.

    Science.gov (United States)

    Fetherolf, Morgan M; Boyd, Stefanie D; Taylor, Alexander B; Kim, Hee Jong; Wohlschlegel, James A; Blackburn, Ninian J; Hart, P John; Winge, Dennis R; Winkler, Duane D

    2017-07-21

    Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs

    International Nuclear Information System (INIS)

    Ravel, P.; Garel, M.C.; Toullec, D.

    1997-01-01

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  17. Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS.

    Science.gov (United States)

    Leitch, Jeffry M; Jensen, Laran T; Bouldin, Samantha D; Outten, Caryn E; Hart, P John; Culotta, Valeria C

    2009-08-14

    Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.

  18. Active colloids

    International Nuclear Information System (INIS)

    Aranson, Igor S

    2013-01-01

    A colloidal suspension is a heterogeneous fluid containing solid microscopic particles. Colloids play an important role in our everyday life, from food and pharmaceutical industries to medicine and nanotechnology. It is useful to distinguish two major classes of colloidal suspensions: equilibrium and active, i.e., maintained out of thermodynamic equilibrium by external electric or magnetic fields, light, chemical reactions, or hydrodynamic shear flow. While the properties of equilibrium colloidal suspensions are fairly well understood, active colloids pose a formidable challenge, and the research is in its early exploratory stage. One of the most remarkable properties of active colloids is the possibility of dynamic self-assembly, a natural tendency of simple building blocks to organize into complex functional architectures. Examples range from tunable, self-healing colloidal crystals and membranes to self-assembled microswimmers and robots. Active colloidal suspensions may exhibit material properties not present in their equilibrium counterparts, e.g., reduced viscosity and enhanced self-diffusivity, etc. This study surveys the most recent developments in the physics of active colloids, both in synthetic and living systems, with the aim of elucidation of the fundamental physical mechanisms governing self-assembly and collective behavior. (physics of our days)

  19. Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

    Science.gov (United States)

    Njuma, Olive J; Davis, Ian; Ndontsa, Elizabeth N; Krewall, Jessica R; Liu, Aimin; Goodwin, Douglas C

    2017-11-10

    KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H 2 O 2 -induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H 2 O 2 to O 2 and H 2 O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H 2 O 2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H 2 O 2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Physics activities

    International Nuclear Information System (INIS)

    1997-09-01

    As we move into the 21st Century, nuclear technology is on the verge of rejuvenation in advanced Member States and of expansion in developing Member States. The principal responsibilities of the IAEA are transferring technologies, co-ordinating scientific research, managing specialized projects and maintaining analytical quality control. The IAEA physics activities provide assistance with nuclear instrumentation, promote more effective utilization of research reactors and accelerators, and facilitate global co-operation in nuclear fusion research. These activities will help Member States improve their standards of living through the benefits of nuclear technology. This booklet presents a brief profile on the physics activities and involvement in these fields of the Physics Section, IAEA

  1. Regulatory activities

    International Nuclear Information System (INIS)

    2001-01-01

    This publication, compiled in 8 chapters, presents the regulatory system developed by the Nuclear Regulatory Authority (NRA) of the Argentine Republic. The following activities and developed topics in this document describe: the evolution of the nuclear regulatory activity in Argentina; the Argentine regulatory system; the nuclear regulatory laws and standards; the inspection and safeguards of nuclear facilities; the emergency systems; the environmental systems; the environmental monitoring; the analysis laboratories on physical and biological dosimetry, prenatal irradiation, internal irradiation, radiation measurements, detection techniques on nuclear testing, medical program on radiation protection; the institutional relations with national and international organization; the training courses and meeting; the technical information

  2. Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals.

    Science.gov (United States)

    Darwish, W; Ikenaka, Y; Eldaly, E; Ishizuka, M

    2010-01-01

    The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using alpha-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  3. Effects of metal-ion replacement on pyrazinamidase activity: A quantum mechanical study.

    Science.gov (United States)

    Khadem-Maaref, Mahmoud; Mehrnejad, Faramarz; Phirouznia, Arash

    2017-05-01

    Pyrazinamidase (PZase), a metalloenzyme, is responsible for acidic modification of pyrazinamide (PZA), a drug used in tuberculosis treatment. The metal coordination site of the enzyme is able to coordinate various divalent metal cofactors. Previous experimental studies have demonstrated that metal ions, such as Co 2+ , Mn 2+ , and Zn 2+ , are able to reactivate metal-depleted PZase, while others including Cu 2+ , Fe 2+ , and Mg 2+ , cannot restore activity. In this study, we investigated binding of various metal ions to the metal coordination site (MCS) of the enzyme using quantum mechanical calculations. We calculated the metal-ligand (residue) binding energy and the atomic partial charges in the presence of various ions. The results indicated that the tendency of alkaline earth metals to bind to PZase MCS is very low and not suitable for enzyme structural and catalytic function. In contrast, Co 2+ and Ni 2+ ions have very high binding affinity and are favorable to the structural and functional properties of the enzyme. Furthermore, we observed that the rate at which Ni 2+ , Co 2+ and Fe 2+ ions in PZase MCS polarize the OH bond of coordinated water molecules is much higher than the polarization rate created by other ions. This finding suggests that the coordination of Ni 2+ , Co 2+ , or Fe 2+ to PZase facilitates the deprotonation of coordinated water molecules to generate a nucleophile that catalyzes the enzymatic reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Inhibition of tryptophan - pyrrolase activity and elevation of brain tryptophan concentration by fluoxetine in rats

    International Nuclear Information System (INIS)

    Bano, S.; Sherkheli, M.A

    2003-01-01

    Objective: To investigate in-vitro as well as in-vivo effects of various doses of fluoxetine (SSRI) on tryptophan metabolism in rates. Results: In in-vitro (10 - 1000 mM) as well in-vivo (0.5 - 30 mg/kg body wt.) studies, fluoxetine showed a statistically significant inhibition of rat liver tryptophan pyrrolase (tryptophan-2,3-dioxygenase; EC 1.13.11.11) activity. Significant increases were noted at 10 and 30 mg/kg doses in brain, serum (total and free) and liver L-tryptophan concentrations. Similarly, serum non-esterified free fatty acids showed a significant increase at both doses. There was no effect on serum glucose and albumin concentrations. Conclusion: It is suggested that major mechanism of action of fluoxetine is that of elevating brain tryptophan concentration and hence 5-HT synthesis by increasing the availability of circulating tryptophan to the brain secondarily to inhibition of major tryptophan degrading enzyme, hepatic tryptophan pyrrolase. It is assumed that fluoxetine inhibits the binding of apoenzyme form of tryptophan pyrrolase with its cofactor haem. The results are discussed in relation to possible involvement of disturbed hepatic tryptophan metabolism in depressive illness. (author)

  5. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Directory of Open Access Journals (Sweden)

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  6. Characterization of the expression, promoter activity and molecular architecture of fibin

    Directory of Open Access Journals (Sweden)

    Hermsdorf Thomas

    2011-05-01

    Full Text Available Abstract Background Fibin was initially discovered as a secreted signal molecule essential for pectoral fin bud initiation in zebrafish. Currently, there is little information about the molecular architecture and biological relevance of fibin in humans and other mammals. Results Fibin is expressed in cerebellum, skeletal muscle and many other embryonic and adult mouse tissues suggesting not only a role during embryonic development but also in adult functions. A 2.5-kbp genomic sequence fragment upstream of the coding sequence is sufficient to drive and regulate fibin expression through stimulation by glucocorticoids, activators of the protein kinase C signalling pathways and manganese ions. Fibin is an evolutionarily conserved protein, carries a cleavable signal peptide (amino acids 1-18 and is glycosylated at Asn30. The two conserved cysteines participate in intermolecular disulfide bond and multimer formation. Although fibin displays all features of a secretory protein, it is mostly retained in the endoplasmic reticulum when heterologously expressed. Conclusion Fibin is functionally relevant during embryogenesis and adult life. Its expression is regulated by a number of cellular signalling pathways and the protein is routed via the secretory pathway. However, proper secretion presumably requires an unknown covalently-linked or associated co-factor.

  7. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.

    Science.gov (United States)

    Preissler, Steffen; Rato, Cláudia; Chen, Ruming; Antrobus, Robin; Ding, Shujing; Fearnley, Ian M; Ron, David

    2015-12-17

    The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

  8. Identity Activities

    Science.gov (United States)

    2016-08-03

    in reaction to their environment. They reflect an individual’s internal or external, conscious or subconscious , overt or covert, voluntary or...identity activities under a range of legal authorities, policy constraints, transnational threats, regional concerns and biases , and most likely...Biography. A baseline and descriptive analytic product that supports the development of the behavioral influences analysis ( BIA ) individual behavioral

  9. Active instruments

    DEFF Research Database (Denmark)

    Lim, Miguel Antonio; Ørberg, Jakob Williams

    2017-01-01

    themselves. We draw on two multi-year field studies of India and Denmark to investigate how national reforms and developments within the ranking industry interact in often surprising ways. Rankings do not always do what policy makers expect. We (1) highlight the activity of rankers in these two countries, (2...

  10. Active house

    DEFF Research Database (Denmark)

    Eriksen, Kurt Emil; Olesen, Gitte Gylling Hammershøj

    Formålet med dette abstrakt er at illustrere, at huse kan være konstrueret til at basere sig udelukkende på vedvarende energikilder og samtidig være CO2-neutrale og producere mere energi end de forbruger. Active House Visionen undersøger disse muligheder i otte demonstration huse i fem forskellige...

  11. Modeling of human factor Va inactivation by activated protein C

    Directory of Open Access Journals (Sweden)

    Bravo Maria

    2012-05-01

    Full Text Available Abstract Background Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (FXa and its cofactor FVa plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC, an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and “protection” of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation. Results A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative

  12. Molecular cloning and characterization of rhesus monkey platelet glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex.

    Science.gov (United States)

    Qiao, Jianlin; Shen, Yang; Shi, Meimei; Lu, Yanrong; Cheng, Jingqiu; Chen, Younan

    2014-05-01

    Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα. DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation. Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα-binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses. Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Pyrroloquinoline quinone (PQQ inhibits lipopolysaccharide induced inflammation in part via downregulated NF-κB and p38/JNK activation in microglial and attenuates microglia activation in lipopolysaccharide treatment mice.

    Directory of Open Access Journals (Sweden)

    Chongfei Yang

    Full Text Available Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. Pyrroloquinoline quinone (PQQ is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and has been reported to exert potent immunosuppressive effects. In the present study, the anti-inflammatory effects of PQQ was investigated in LPS treated primary microglia cells. Our observations showed that pretreatment with PQQ significantly inhibited the production of NO and PGE2 and suppressed the expression of pro-inflammatory mediators such as iNOS, COX-2, TNF-a, IL-1b, IL-6, MCP-1 and MIP-1a in LPS treated primary microglia cells. The nuclear translocation of NF-κB and the phosphorylation level of p65, p38 and JNK MAP kinase pathways were also inhibited by PQQ in LPS stimulated primary microglia cells. Further a systemic LPS treatment acute inflammation murine brain model was used to study the suppressive effects of PQQ against neuroinflammation in vivo. Mice treated with PQQ demonstrated marked attenuation of neuroinflammation based on Western blotting and immunohistochemistry analysis of Iba1-against antibody in the brain tissue. Indicated that PQQ protected primary cortical neurons against microglia-mediated neurotoxicity. These results collectively suggested that PQQ might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.

  14. Active particles

    CERN Document Server

    Degond, Pierre; Tadmor, Eitan

    2017-01-01

    This volume collects ten surveys on the modeling, simulation, and applications of active particles using methods ranging from mathematical kinetic theory to nonequilibrium statistical mechanics. The contributing authors are leading experts working in this challenging field, and each of their chapters provides a review of the most recent results in their areas and looks ahead to future research directions. The approaches to studying active matter are presented here from many different perspectives, such as individual-based models, evolutionary games, Brownian motion, and continuum theories, as well as various combinations of these. Applications covered include biological network formation and network theory; opinion formation and social systems; control theory of sparse systems; theory and applications of mean field games; population learning; dynamics of flocking systems; vehicular traffic flow; and stochastic particles and mean field approximation. Mathematicians and other members of the scientific commu...

  15. Active solar information dissemination activities

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-07-01

    The principal objective of the project has been the development of an information dissemination strategy for the UK active solar heating industry. The project has also aimed to prepare the industry for the implementation of such a strategy and to produce initial information materials to support the early stages of the implementation process. (author)

  16. Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

    Science.gov (United States)

    Dobisova, Tereza; Hrdinova, Vendula; Cuesta, Candela; Michlickova, Sarka; Urbankova, Ivana; Hejatkova, Romana; Zadnikova, Petra; Pernisova, Marketa; Benkova, Eva; Hejatko, Jan

    2017-05-01

    In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL ( LPH ) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 ( CKI1 ), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 ( HY1 ) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph / hy1 - 7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  17. c-Abl phosphorylation of Yin Yang 1's conserved tyrosine 254 in the spacer region modulates its transcriptional activity.

    Science.gov (United States)

    Daraiseh, Susan I; Kassardjian, Ari; Alexander, Karen E; Rizkallah, Raed; Hurt, Myra M

    2018-05-25

    Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways. Copyright © 2018. Published by Elsevier B.V.

  18. Alpha 1,3-Galactosyltransferase Deficiency in Pigs Increases Sialyltransferase Activities That Potentially Raise Non-Gal Xenoantigenicity

    Directory of Open Access Journals (Sweden)

    Jong-Yi Park

    2011-01-01

    Full Text Available We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST and α2,6-sialyltransferase (α2,6ST] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH, which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

  19. Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

    Science.gov (United States)

    Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

    2016-05-01

    Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Synthesis, in vitro and in silico antimalarial activity of 7-chloroquinoline and 4H-chromene conjugates.

    Science.gov (United States)

    Parthiban, A; Muthukumaran, J; Manhas, Ashan; Srivastava, Kumkum; Krishna, R; Rao, H Surya Prakash

    2015-10-15

    A new series of chloroquinoline-4H-chromene conjugates incorporating piperizine or azipane tethers were synthesized and their anti-malarial activity were evaluated against two Plasmodium falciparum strains namely 3D7 chloroquine sensitive (CQS) and K1 chloroquine resistant (CQR). Chloroquine was used as the standard and also reference for comparison. The conjugates exhibit intense UV absorption with λmax located at 342 nm (log ε=4.0), 254 nm (log ε=4.2), 223 nm (log ε=4.4) which can be used to spectrometrically track the molecules even in trace amounts. Among all the synthetic compounds, two molecules namely 6-nitro and N-piperazine groups incorporated 7d and 6-chloro and N-azapane incorporated 15b chloroquinoline-4H-chromene conjugates showed significant anti-malarial activity against two strains (3D7 and K1) of P. falciparum. These values are lesser than the values of standard antimalarial compound. Molecular docking results suggested that these two compounds showing strong binding affinity with P. falciparum lactate dehydrogenase (PfLDH) and also they occupy the co-factor position which indicated that they could be the potent inhibitors for dreadful disease malaria and specifically attack the glycolytic pathway in parasite for energy production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Mangiferin Upregulates Glyoxalase 1 Through Activation of Nrf2/ARE Signaling in Central Neurons Cultured with High Glucose.

    Science.gov (United States)

    Liu, Yao-Wu; Cheng, Ya-Qin; Liu, Xiao-Li; Hao, Yun-Chao; Li, Yu; Zhu, Xia; Zhang, Fan; Yin, Xiao-Xing

    2017-08-01

    Mangiferin, a natural C-glucoside xanthone, has anti-inflammatory, anti-oxidative, neuroprotective actions. Our previous study showed that mangiferin could attenuate diabetes-associated cognitive impairment of rats by enhancing the function of glyoxalase 1 (Glo-1) in brain. The aim of this study was to investigate whether Glo-1 upregulation by mangiferin in central neurons exposed to chronic high glucose may be related to activation of Nrf2/ARE pathway. Compared with normal glucose (25 mmol/L) culture, Glo-1 protein, mRNA, and activity levels were markedly decreased in primary hippocampal and cerebral cortical neurons cultured with high glucose (50 mmol/L) for 72 h, accompanied by the declined Nrf2 nuclear translocation and protein expression of Nrf2 in cell nucleus, as well as protein expression and mRNA level of γ-glutamylcysteine synthetase (γ-GCS) and superoxide dismutase activity, target genes of Nrf2/ARE signaling. Nonetheless, high glucose cotreating with mangiferin or sulforaphane, a typical inducer of Nrf2 activation, attenuated the above changes in both central neurons. In addition, mangiferin and sulforaphane significantly prevented the formation of advanced glycation end-products (AGEs) reflecting Glo-1 activity, while elevated the level of glutathione, a cofactor of Glo-1 activity and production of γ-GCS, in high glucose cultured central neurons. These findings demonstrated that Glo-1 was greatly downregulated in central neurons exposed to chronic high glucose, which is expected to lead the formation of AGEs and oxidative stress damages. We also proved that mangiferin enhanced the function of Glo-1 under high glucose condition by inducing activation of Nrf2/ARE signaling pathway.

  2. Chocolate active

    International Nuclear Information System (INIS)

    Anon.

    1987-01-01

    There is a table of current radioactivity values for various foods and mushrooms. A special accent is on milk and chocolate. Chocolate sorts with more powdered milk are more active. Finally there is a chapter on radionucleides contained in the Chernobyl fallout, other than cesium 137, cesium 134 and strontium 90. The amounts of ruthenium 106, antimony 125, cerium 144, silver 110 m, cesium 134, strontium 90 and plutonium 239 relative to cesium 137 in soil samples in autumn 1987 are given. Special emphasis is on ruthenium 'hot particles' and on plutonium. (qui)

  3. Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Murugesan Velayutham

    2016-03-01

    Full Text Available In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+cyt c in the absence and presence of H2O2. Electron paramagnetic resonance (EPR spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO were performed with sulfite, Fe3+cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3- was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which

  4. Effect of human cell malignancy on activity of DNA polymerase iota.

    Science.gov (United States)

    Kazakov, A A; Grishina, E E; Tarantul, V Z; Gening, L V

    2010-07-01

    An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by

  5. Staying Active: Physical Activity and Exercise

    Science.gov (United States)

    ... Events Advocacy For Patients About ACOG Staying Active: Physical Activity and Exercise Home For Patients Search FAQs Staying ... Exercise FAQ045, November 2016 PDF Format Staying Active: Physical Activity and Exercise Women's Health What are the benefits ...

  6. Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS.

    Science.gov (United States)

    Jensen, Laran T; Culotta, Valeria Cizewski

    2005-12-16

    Reactive oxygen species are produced as the direct result of aerobic metabolism and can cause damage to DNA, proteins, and lipids. A principal defense against reactive oxygen species involves the superoxide dismutases (SOD) that act to detoxify superoxide anions. Activation of CuZn-SODs in eukaryotic cells occurs post-translationally and is generally dependent on the copper chaperone for SOD1 (CCS), which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. In contrast to other eukaryotes, the nematode Caenorhabditis elegans does not contain an obvious CCS homologue, and we have found that the C. elegans intracellular CuZn-SODs (wSOD-1 and wSOD-5) are not dependent on CCS for activation when expressed in Saccharomyces cerevisiae. CCS-independent activation of CuZn-SODs is not unique to C. elegans; however, this is the first organism identified that appears to exclusively use this alternative pathway. As was found for mammalian SOD1, wSOD-1 exhibits a requirement for reduced glutathione in CCS-independent activation. Unexpectedly, wSOD-1 was inactive even in the presence of CCS when glutathione was depleted. Our investigation of the cysteine residues that form the disulfide bond in wSOD-1 suggests that the ability of wSODs to readily form this disulfide bond may be the key to obtaining high levels of activation through the CCS-independent pathway. Overall, these studies demonstrate that the CuZn-SODs of C. elegans have uniquely evolved to acquire copper without the copper chaperone and this may reflect the lifestyle of this organism.

  7. Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

    International Nuclear Information System (INIS)

    Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

    2015-01-01

    Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis

  8. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

    2011-01-31

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  9. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  10. Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats.

    Directory of Open Access Journals (Sweden)

    Nady Braidy

    2011-04-01

    Full Text Available The cofactor nicotinamide adenine dinucleotide (NAD+ has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose polymerase (PARP, an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months compared to young (i.e. 3 month old rats. These changes in [NAD(H] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

  11. Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

    Directory of Open Access Journals (Sweden)

    Gerasimos F Kremmydas

    Full Text Available Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ, and two genes (sup5 and sup6 which seem to be organized in a putative operon. This operon (named supX consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

  12. Human activated protein C variants in a rat model of arterial thrombosis

    Directory of Open Access Journals (Sweden)

    Dahlbäck Björn

    2008-10-01

    Full Text Available Abstract Background Activated protein C (APC inhibits coagulation by degrading activated factor V (FVa and factor VIII (FVIIIa, protein S (PS functioning as a cofactor to APC. Methods By mutagenesis of the vitamin K-dependent Gla domain of APC, we have recently created an APC variant having enhanced anticoagulant activity due to increased affinity for negatively charged phospholipid membranes. In the present study, the potential antithrombotic effects of this APC variant, and of a variant APC that is additionally mutated in the serine protease domain, have been evaluated in a blind randomized study in a rat model of arterial thrombosis. In this model, we have previously found the combination of bovine APC and PS to be highly antithrombotic. Four treatment groups each containing 10 rats were, in a blind random fashion, given intravenous bolus injections of wild-type or mutant variants of APC (0.8 mg/kg together with human PS (0.6 mg/kg or human PS (0.6 mg/kg alone. A control group with 20 animals where given vehicle only. Results A trend to increased patency rates was noted in a group receiving one of the APC variants, but it did not reach statistical significance. Conclusion In conclusion, administration of human APC variants having enhanced anticoagulant efficacy together with human PS in a rat model of arterial thrombosis did not give an efficient antithrombotic effect. The lack of effect may be due to species-specific differences between the human protein C system and the rat hemostatic system.

  13. Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

    Science.gov (United States)

    Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

    2014-07-23

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  14. Halal Activism

    DEFF Research Database (Denmark)

    Fischer, Johan

    2016-01-01

    The purpose of this article is to further our understanding of contemporary Muslim consumer activism in Malaysia with a particular focus on halal (in Arabic, literally “permissible” or “lawful”) products and services. Muslim activists and organisations promote halal on a big scale in the interface...... zones between new forms of Islamic revivalism, the ethnicised state and Muslim consumer culture. Organisations such as the Muslim Consumers Association of Malaysia play an important role in pushing and protecting halal in Malaysia, that is, halal activists constantly call on the state to tighten halal...... in particular historical/national settings and that these issues should be explored in the interfaces between Islam, the state and market. More specifically, this article examines the above issues building on ethnography from fieldwork with three Muslim organisations in Malaysia....

  15. Structural Analysis and Anticoagulant Activities of the Novel Sulfated Fucan Possessing a Regular Well-Defined Repeating Unit from Sea Cucumber

    Directory of Open Access Journals (Sweden)

    Mingyi Wu

    2015-04-01

    Full Text Available Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC–MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1→2 and (1→3-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.

  16. The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity.

    Directory of Open Access Journals (Sweden)

    Cendrine Tourette

    2014-06-01

    Full Text Available The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD. Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT in several models of Huntington's disease (HD. Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor β-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished β-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.

  17. The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

    Science.gov (United States)

    Karmohapatra, Soumendra K; Chakraborty, Kushal; Kahn, Nighat N; Sinha, Asru K

    2007-11-01

    Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture. (c) 2007 Wiley-Liss, Inc.

  18. Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

    Science.gov (United States)

    Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

    2014-02-05

    A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

  19. Active sharing

    CERN Multimedia

    2012-01-01

    The big news this week is, of course, the conclusions from the LHC performance workshop held in Chamonix from 6 to 10 February . The main recommendation, endorsed by CERN’s Machine Advisory Committee and adopted by the Management, is that the LHC will run at 4 TeV per beam this year. You can find all the details from Chamonix in the slides presented on Wednesday at the summary session, which leaves me free to talk about another important development coming up soon.   In ten days time, a new kind of gathering will be taking place in Geneva, bringing together two previously separate conferences, one driven by physics, the other by the medical community, but both looking to apply physics to the advancement of health. The merger of the International Conference for Translational Research in Radio-Oncology and CERN’s workshop on Physics for Health in Europe (ICTR-PHE) makes for a very eclectic mix. Presentations range from active shielding for interplanetary flight to the rather...

  20. Active Segmentation.

    Science.gov (United States)

    Mishra, Ajay; Aloimonos, Yiannis

    2009-01-01

    The human visual system observes and understands a scene/image by making a series of fixations. Every fixation point lies inside a particular region of arbitrary shape and size in the scene which can either be an object or just a part of it. We define as a basic segmentation problem the task of segmenting that region containing the fixation point. Segmenting the region containing the fixation is equivalent to finding the enclosing contour- a connected set of boundary edge fragments in the edge map of the scene - around the fixation. This enclosing contour should be a depth boundary.We present here a novel algorithm that finds this bounding contour and achieves the segmentation of one object, given the fixation. The proposed segmentation framework combines monocular cues (color/intensity/texture) with stereo and/or motion, in a cue independent manner. The semantic robots of the immediate future will be able to use this algorithm to automatically find objects in any environment. The capability of automatically segmenting objects in their visual field can bring the visual processing to the next level. Our approach is different from current approaches. While existing work attempts to segment the whole scene at once into many areas, we segment only one image region, specifically the one containing the fixation point. Experiments with real imagery collected by our active robot and from the known databases 1 demonstrate the promise of the approach.

  1. IASS Activity

    Science.gov (United States)

    Hojaev, Alisher S.; Ibragimova, Elvira M.

    2015-08-01

    It’s well known, astronomy in Uzbekistan has ancient roots and traditions (e.g., Mirzo Ulugh Beg, Abū al-Rayhān al-Bīrūnī, Abū ‘Abdallāh al-Khwārizmī) and astronomical heritage carefully preserved. Nowadays uzbek astronomers play a key role in scientific research but also in OAD and Decadal Plan activity in the Central Asia region. International Aerospace School (IASS) is an amazing and wonderful event held annually about 30 years. IASS is unique project in the region, and at the beginning we spent the Summer and Winter Schools. At present in the summer camp we gather about 50 teenage and undergraduate students over the country and abroad (France, Malaysia, Turkey, Azerbaijan, Pakistan, Russia, etc.). They are selected on the basis of tests of astronomy and space issues. During two weeks of IASS camp the invited scientists, cosmonauts and astronauts as well as other specialists give lectures and engage in practical exercises with IASS students in astronomy, including daily observations of the Sun and night sky observations with meniscus telescope, space research and exploration, aerospace modelling, preparation and presentation of original projects. This is important that IASS gives not theoretical grounds only but also practically train the students and the hands-on training is the major aims of IASS. Lectures and practice in the field of astronomy carried out with the direct involvement and generous assistance of Uranoscope Association (Paris, France). The current 26-th IASS is planned to held in July 2015.

  2. Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland.

    Science.gov (United States)

    Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Borgatti, Anna Rosa; Pagliarani, Alessandra

    2011-02-01

    Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  4. Activation Energy

    Science.gov (United States)

    Gadeken, Owen

    2002-01-01

    Teaming is so common in today's project management environment that most of us assume it comes naturally. We further assume that when presented with meaningful and challenging work, project teams will naturally engage in productive activity to complete their tasks. This assumption is expressed in the simple (but false) equation: Team + Work = Teamwork. Although this equation appears simple and straightforward, it is far from true for most project organizations whose reality is a complex web of institutional norms based on individual achievement and rewards. This is illustrated by the very first successful team experience from my early Air Force career. As a young lieutenant, I was sent to Squadron Officer School, which was the first in the series of Air Force professional military education courses I was required to complete during my career. We were immediately formed into teams of twelve officers. Much of the course featured competition between these teams. As the most junior member of my team, I quickly observed the tremendous pressure to show individual leadership capability. At one point early in the course, almost everyone in our group was vying to become the team leader. This conflict was so intense that it caused us to fail miserably in our first outdoor team building exercise. We spent so much time fighting over leadership that we were unable to complete any of the events on the outdoor obstacle course. This complete lack of success was so disheartening to me that I gave our team little hope for future success. What followed was a very intense period of bickering, conflict, and even shouting matches as our dysfunctional team tried to cope with our early failures and find some way to succeed. British physician and researcher Wilfred Bion (Experiences in Groups, 1961) discovered that there are powerful psychological forces inherent in all groups that divert from accomplishing their primary tasks. To overcome these restraining forces and use the potential

  5. Antiphospholipid and antioangiogenic activity in females with recurrent miscarriage and antiphospholipid syndrome.

    Science.gov (United States)

    Pelusa, Hector F; Pezzarini, Eleonora; Basiglio, Cecilia L; Musuruana, Jorge; Bearzotti, Mariela; Svetaz, María J; Daniele, Stella M; Bottai, Hebe; Arriaga, Sandra Mm

    2017-09-01

    Background Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis, fetal losses and thrombocytopenia associated to antiphospholipid antibodies. They are directed to phospholipids, such as cardiolipins (anticardiolipin) and lupus anticoagulant or to complexes formed by phospholipids and protein cofactors, such as β2 glycoprotein 1 (a-β2GP1) and annexin V (a-annexin V). These auto-antibodies may be considered as a family of antibodies involved in thrombotic events and antiphospholipid activity. On the other hand, some proangiogenic factors are involved in the normal development of placental vasculature, such as the vascular endothelial growth factor. Overexpression of vascular endothelial growth factor receptor in its soluble form (sVEGFR-1) has been associated to a higher antiangiogenic activity. Our aim was to analyse the association between anticardiolipin, lupus anticoagulant, a-β2GP1, a-annexin V and sVEGFR-1 with recurrent miscarriage before week 10 of gestation in females with antiphospholipid syndrome. Methods We studied 24 females (primary or secondary antiphospholipid syndrome), who were divided into two groups: females with recurrent miscarriage before week 10 of gestation (M; n = 12) and females with no history of fetal loss (NM; n = 12). Anticardiolipin, a-β2GP1, a-annexin V and sVEGF-R1 concentrations were assessed by ELISA, while lupus anticoagulant was assessed by screening and confirmatory tests. Results A significant association was observed between the number of positive biomarkers and the belonging group ( P antiphospholipid syndrome.

  6. ENDOTHELIUM LESION MARKERS AND THROMBOCYTE AGGREGATION IN CHRONIC HEPATITIS AND HEPATIC CIRRHOSIS

    Directory of Open Access Journals (Sweden)

    A. P. Shchekotova

    2012-01-01

    Full Text Available Aim — to estimate endothelium lesion, quantity and thrombocyte aggregation function correlation in viral chronic hepatitis C (CHC and hepatic cirrhosis (HC.Materials and methods. 50 CHC patients and 28 HC patients were examined. Using IFA method the total nitric oxide, endothelin‑1, vasculoendothelial growth factor levels, Willebrand factor (vWF activity were investigated, blood plasma desquamated endotheliocyte (DEC number was calculated with Hladovec method, 1978, thrombocyte aggregation (TA with ADP, collagen, ristocetine was determined.Results. DEC and vWF demonstrated correlation in CHC (p = 0.014 and HC (p = 0.000004. In HC patients reliable correlation of all the investigated indices of endothelium lesion with the thrombocyte number and TA was detected, but in CHC patients no correlations were revealed. Thus, significant elevation of TA with ristocetine was noted only in CHC. Decrease in thrombocyte amount among CHC patients and,especially in HC, and heightened vWF activity could change true TA indices. The corrected TA, whose indices in hepatic diseases significantlyincreased, was calculated taking into account the correction factor vWF / thrombocytes that in CHC did not differ from that of healthy patients and in HC was essentially higher.Conclusion. Endothelium dysfunction markers in CH and HC demonstrate correlation with thrombocyte reduction and TA elevation. Determinationof corrected TA permits to reveal disturbances of thrombocyte hemostasis in the form of elevated aggregation in all CHC and HC patients.

  7. High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

    Directory of Open Access Journals (Sweden)

    Grinsztejn Beatriz

    2010-12-01

    Full Text Available Abstract Background Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL and tegumentary leishmaniasis (ATL have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods To address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3. Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

  8. Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

    Science.gov (United States)

    Panagos, Charalampos G; Thomson, Derek S; Moss, Claire; Hughes, Adam D; Kelly, Maeve S; Liu, Yan; Chai, Wengang; Venkatasamy, Radhakrishnan; Spina, Domenico; Page, Clive P; Hogwood, John; Woods, Robert J; Mulloy, Barbara; Bavington, Charlie D; Uhrín, Dušan

    2014-10-10

    Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcβ4,6S(1 → 4) [FucαX(1 → 3)]GlcAβ(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Role of metabolic activation by cytochrome P-450 in covalent binding of VP 16-213 to rat liver and HeLa cell microsomal proteins

    Energy Technology Data Exchange (ETDEWEB)

    van Maanen, J.M.; de Ruiter, C.; de Vries, J.; Kootstra, P.R.; Gobas, F.; Pinedo, H.M.

    1985-09-01

    Covalent binding of /sup 3/H-labeled VP 16-213 to rat liver and HeLa cell microsomal proteins was studied in vitro. Metabolic activation by cytochrome P-450 was found to play a role in the covalent binding of VP 16-213 to rat liver microsomal proteins, as shown by the need of NADPH cofactor, the increased binding after phenobarbital pretreatment and the inhibition by SFK-525A. Addition of ascorbic acid or alpha-phenyl-N-tert. butylnitrone to the incubation mixture depressed covalent binding by about 85%, suggesting that formation of a reactive metabolite from the phenolic structure may be involved in the binding process. VP 16-213 did not inhibit aminopyrine N-demethylase at the concentration used in the binding experiments (17 microM), indicating that metabolism of its methylenedioxy group does not play a role in binding to microsomal proteins. HeLa cell microsomes were found to possess aminopyrine N-demethylase activity. Covalent binding of radiolabeled VP 16-213 to HeLa cell microsomes decreased by about 64% if NADPH was omitted.

  10. SIRT3 and SIRT5 regulate the enzyme activity and cardiolipin binding of very long-chain acyl-CoA dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Yuxun Zhang

    Full Text Available SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD, a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.

  11. S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site.

    Science.gov (United States)

    Kanda, Hironori; Toyama, Takashi; Shinohara-Kanda, Azusa; Iwamatsu, Akihiro; Shinkai, Yasuhiro; Kaji, Toshiyuki; Kikushima, Makoto; Kumagai, Yoshito

    2012-11-01

    We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

  12. Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

    International Nuclear Information System (INIS)

    Zhao Cunyou; Chen Yali; Park, Jiyoung; Kim, Jae Bum; Tang Hong

    2004-01-01

    Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication

  13. Measuring Physical Activity Intensity

    Medline Plus

    Full Text Available ... Needs for Pregnant or Postpartum Women Physical Activity & Health Adding Physical Activity to Your Life Activities for ... Guide Visual Guide Worksite Physical Activity Steps to Wellness Walkability Audit Tool Sample Audit Glossary Selected References ...

  14. Measuring Physical Activity Intensity

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    Full Text Available ... for Older Adults Needs for Pregnant or Postpartum Women Physical Activity & Health Adding Physical Activity to Your ... Activity, 2014 Recommendations & Guidelines Fact Sheets & Infographics Social Media Tools Community Strategies BE Active: Connecting Routes + Destinations ...

  15. Measuring Physical Activity Intensity

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    Full Text Available ... Physical Activity Data, Trends and Maps Surveillance Systems Resources & Publications Reports Adults Need More Physical Activity MMWR ... Active: Connecting Routes + Destinations Real-World Examples Implementation Resource Guide Visual Guide Worksite Physical Activity Steps to ...

  16. Measuring Physical Activity Intensity

    Medline Plus

    Full Text Available ... Physical Activity Basics Needs for Adults Needs for Children What Counts Needs for Older Adults Needs for ... Adding Physical Activity to Your Life Activities for Children Activities for Older Adults Overcoming Barriers Measuring Physical ...

  17. Measuring Physical Activity Intensity

    Medline Plus

    Full Text Available ... to Your Life Activities for Children Activities for Older Adults Overcoming Barriers ... required by a person to do an activity. When using relative intensity, people pay attention to how physical activity affects their ...

  18. Measuring Physical Activity Intensity

    Medline Plus

    Full Text Available ... Adults Need More Physical Activity MMWR Data Highlights State Indicator Report on Physical Activity, 2014 Recommendations & Guidelines ... Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs Measuring Physical Activity Intensity Recommend ...

  19. Measuring Physical Activity Intensity

    Medline Plus

    Full Text Available ... Adults Needs for Children What Counts Needs for Older Adults Needs for Pregnant or Postpartum Women Physical Activity & ... to Your Life Activities for Children Activities for Older Adults Overcoming Barriers Measuring Physical Activity Intensity Target Heart ...

  20. BAM! Physical Activity

    Science.gov (United States)

    ... Smarts Links Fuel Up for Fun Power Packing Physical Activity Activity Calendar Activity Information Sheets I Heard Hurdle ... Links Sleep Game Questions Answered Under the Microscope Physical Activity Game Questions Answered Under the Microscope Lurking in ...

  1. Facts about Physical Activity

    Science.gov (United States)

    ... Micronutrient Malnutrition State and Local Programs Facts about Physical Activity Recommend on Facebook Tweet Share Compartir Some Americans ... Activity Guideline for aerobic activity than older adults. Physical activity and socioeconomic status Adults with more education are ...

  2. Physical Activity Guidelines

    Science.gov (United States)

    ... use this site. health.gov Physical Activity Guidelines Physical Activity Physical activity is key to improving the health of the Nation. Based on the latest science, the Physical Activity Guidelines for Americans is an essential resource for ...

  3. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xu-Dong [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Meng, Qing-Hui [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Xu, Jia-Ying; Jiao, Yang [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Ge, Chun-Min [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Jacob, Asha; Wang, Ping [North Shore University Hospital-Long Island Jewish Medical Center and The Feinstein Institute for Medical Research, Manhasset, NY 11030 (United States); Rosen, Eliot M [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Fan, Saijun, E-mail: sjfan@suda.edu.cn [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China)

    2011-01-28

    Research highlights: {yields} BTG2 associates with AR, androgen causes an increase of the interaction. {yields} BTG2 as a co-repressor inhibits the AR-mediated transcription activity. {yields} BTG2 inhibits the transcription activity and expression of PSA. {yields} An intact {sup 92}LxxLL{sup 96} motif is essential and necessary for these activities