Science.gov

Sample records for 4-oxalocrotonate tautomerase 4-ot

  1. Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1

    PubMed Central

    Terrell, Cassidy R.; Burks, Elizabeth A.; Whitman, Christian P.; Hoffman, David W.

    2013-01-01

    Methylibium petroleiphilumstrain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwise sequence identity (35%) among the isozyme pairs in the parallel pathways, and could offer insight into substrate preferences and pathway functions. The kinetic parameters of tautomerase I and tautomerase II were determined using 2-hydroxymuconate and 5-(methyl)-2-hydroxymuconate. Both tautomerase I and tautomerase II process the substrates, but with different efficiencies. Crystal structures were determined for both tautomerase I and tautomerase II, at 1.57 and 1.64 Å resolution, respectively. The backbones of tautomerase I and tautomerase II are highly similar, but have distinct active site environments. The results, in combination with those for other structurally and kinetically characterized 4-OT isozymes, suggest that tautomerase I catalyzes the tautomerization of both 2-hydroxymuconate and alkyl derivatives, whereas tautomerase II might specialize in other aromatic hydrocarbon metabolites. PMID:23831510

  2. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase: synthesis and evaluation of 2-fluoro substrate analogues.

    PubMed

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-08-17

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with K(i) values ranging from 5- to 633-fold lower than those determined for 4-OT. The study of VPH is complicated by the fact that the enzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD), the enzyme following 4-OT in the catechol meta-fission pathway. A structure-based sequence analysis identified 4-OD as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicated Glu-109 and Glu-111 as potential metal-binding ligands. Changing these residues to a glutamine verified their importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or E111Q4-OD/VPH complexes, which retained full hydratase activity but had little decarboxylase activity. Subsequent incubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomers of the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors. The combined results set the stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands.

  3. Evidence for the formation of an enamine species during aldol and Michael-type addition reactions promiscuously catalyzed by 4-oxalocrotonate tautomerase.

    PubMed

    Poddar, Harshwardhan; Rahimi, Mehran; Geertsema, Edzard M; Thunnissen, Andy-Mark W H; Poelarends, Gerrit J

    2015-03-23

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which has a catalytic N-terminal proline residue (Pro1), can promiscuously catalyze various carbon-carbon bond-forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael-type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ-nitroaldehydes. To gain insight into how 4-OT catalyzes these unnatural reactions, we carried out exchange studies in D2 O, and X-ray crystallography studies. The former established that H-D exchange within acetaldehyde is catalyzed by 4-OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4-OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4-OT-catalyzed aldol and Michael-type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1-dependent formation of an enamine intermediate.

  4. Demethionylation of Pro-1 variants of 4-oxalocrotonate tautomerase in Escherichia coli by co-expression with an engineered methionine aminopeptidase

    PubMed Central

    Baas, Bert-Jan; Zandvoort, Ellen; Wasiel, Anna A.; Poelarends, Gerrit J.

    2014-01-01

    4-Oxalocrotonate tautomerase (4-OT) catalyzes the enol-keto tautomerization of 2-hydroxymuconate, utilizing its N-terminal proline (Pro-1) as general base catalyst. Substituting Pro-1 with bulky or charged residues will result in poor or no post-translational removal of the translation-initiating methionine by the methionine aminopeptidase (MetAP) of the Escherichiacoli expression host. Here, we set out to investigate whether co-expression with previously engineered aminopeptidase MetAP-∗TG can be used to produce the P1S, P1H and P1Q variants of 4-OT in a demethionylated form. The P1S variant, which carries a small residue at the penultimate position (the first position after the initiating methionine), was found to be fully processed by wild-type MetAP. The P1S variant has low-level 2-hydroxymuconate tautomerase and promiscuous oxaloacetate decarboxylase activity. The P1Q and P1H variants of 4-OT, which carry bulky residues at the penultimate position, could only be obtained in a demethionylated form (a minor fraction of the purified protein is still composed of methionylated enzyme) by co-expression with MetAP-∗TG. Interestingly, the Gln-1 residue of the demethionylated P1Q variant undergoes intramolecular cyclization to form pyroglutamate (pE), yielding variant P1pE. Whereas the P1H/M1P2H mixture has low-level tautomerase activity, the P1pE/M1P2Q mixture has robust tautomerase activity. The substitution of Pro-1 by Gln, followed by removal of the initiating Met and cyclization of Gln-1 to form pE, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT. This opens up new possibilities to study the importance of Pro-1 in recently discovered C–C bond-forming activities of this highly promiscuous tautomerase. PMID:25161874

  5. Highly efficient Michael-type addition of acetaldehyde to β-nitrostyrenes by whole resting cells of Escherichia coli expressing 4-oxalocrotonate tautomerase.

    PubMed

    Narancic, Tanja; Radivojevic, Jelena; Jovanovic, Predrag; Francuski, Djordje; Bigovic, Miljan; Maslak, Veselin; Savic, Vladimir; Vasiljevic, Branka; O'Connor, Kevin E; Nikodinovic-Runic, Jasmina

    2013-08-01

    A novel whole cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the asymmetric Michael addition of acetaldehyde to β-nitrostyrenes. Optimal ratio of substrates (2mM β-nitrostyrenes and 20mM acetaldehyde) and biocatalyst of 5 g of cell dry weight of biocatalyst per liter was determined. Through further bioprocess improvement by sequential addition of substrate 10mM nitrostyrene biotransformation was achieved within 150 min. Excellent enantioselectivity (>99% ee) and product yields of up to 60% were obtained with β-nitrostyrene substrate. The biotransformation product, 4-nitro-3-phenyl-butanal, was isolated from aqueous media and further transformed into the corresponding amino alcohol. The biocatalyst exhibited lower reaction rates with p-Cl-, o-Cl- and p-F-β-nitrostyrenes with product yields of 38%, 51%, 31% and ee values of 84%, 88% and 94% respectively. The importance of the terminal proline of 4-OT was confirmed by two proline enriched variants and homology modeling.

  6. 4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy.

    PubMed Central

    Stivers, J. T.; Abeygunawardana, C.; Whitman, C. P.; Mildvan, A. S.

    1996-01-01

    4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta 1, residues 2-8; beta 2, residues 39-45), a beta-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The beta 1 strand is parallel to the beta 2 strand of the same subunit on the basis of cross stand NH(i)-NH(j) NOEs in a 2D 15N-edited 1H-NOESY spectrum of hexameric 4-OT containing two 15N-labeled subunits/hexamer. The beta 1 strand is also antiparallel to another beta 1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the beta-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied. PMID:8845763

  7. Identification and Characterization of New Family Members in the Tautomerase Superfamily: Analysis and Implications

    PubMed Central

    Huddleston, Jamison P.; Burks, Elizabeth A.; Whitman, Christian P.

    2014-01-01

    Tautomerase superfamily members are characterized by a β–α–β building block and a catalytic amino terminal proline. 4-oxalocrotonate tautomerase (4-OT) and malonate semialdehyde decarboxylase (MSAD) are the title enzymes of two of the five known families in the superfamily. Two recent developments in these families indicate that there might be more metabolic diversity in the tautomerase superfamily than previously thought. 4-OT homologues have been identified in three biosynthetic pathways, whereas all previously characterized 4-OTs are found in catabolic pathways. In the MSAD family, homologues have been characterized that lack decarboxylase activity, but have a modest hydratase activity using 2-oxo-3-pentynoate. This observation stands in contrast to the first characterized MSAD, which is a proficient decarboxylase and a less efficient hydratase. The hydratase activity was thought to be a vestigial and promiscuous activity. However, this recent discovery suggests that the hydratase activity might reflect a new activity in the MSAD family for an unknown substrate. These discoveries open up new avenues of research in the tautomerase superfamily. PMID:25219626

  8. Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases

    PubMed Central

    van der Meer, Jan-Ytzen; Poddar, Harshwardhan; Baas, Bert-Jan; Miao, Yufeng; Rahimi, Mehran; Kunzendorf, Andreas; van Merkerk, Ronald; Tepper, Pieter G.; Geertsema, Edzard M.; Thunnissen, Andy-Mark W. H.; Quax, Wim J.; Poelarends, Gerrit J.

    2016-01-01

    The Michael-type addition reaction is widely used in organic synthesis for carbon–carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon–carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon–carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence–function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary ‘Michaelases'. These ‘Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives. PMID:26952338

  9. Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.

    PubMed

    Stanley, T M; Johnson, W H; Burks, E A; Whitman, C P; Hwang, C C; Cook, P F

    2000-02-01

    4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.

  10. Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

    PubMed

    Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

    2016-05-10

    The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

  11. Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

    PubMed

    Fitzgerald, M C; Chernushevich, I; Standing, K G; Whitman, C P; Kent, S B

    1996-07-09

    Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

  12. Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

    PubMed Central

    Fitzgerald, M C; Chernushevich, I; Standing, K G; Whitman, C P; Kent, S B

    1996-01-01

    Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT. PMID:8692908

  13. Stereochemical Control of Enzymatic Carbon-Carbon Bond-Forming Michael-Type Additions by "Substrate Engineering".

    PubMed

    Miao, Yufeng; Tepper, Pieter G; Geertsema, Edzard M; Poelarends, Gerrit J

    2016-11-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to β-nitrostyrene derivatives to yield chiral γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acids. In this study, we investigated the effect of different substituents at the aromatic ring of the Michael acceptor on the catalytic efficiency and stereoselectivity of the 4-OT-catalyzed acetaldehyde addition reactions. Highly enantioenriched (R)- and (S)-γ-nitroaldehydes and 4-substituted chroman-2-ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate. Stereochemical control of these enzymatic Michael-type additions by "substrate engineering" allowed the enantioselective synthesis of valuable γ-aminobutyric acid precursors. In addition, the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds for preparing biologically active natural products.

  14. Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity.

    PubMed

    Tarasuk, Mayuri; Poungpair, Ornnuthchar; Ungsupravate, Duangporn; Bangphoomi, Kunan; Chaicumpa, Wanpen; Yenchitsomanus, Pa-Thai

    2014-03-01

    Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

  15. Discovery of covalent inhibitors for MIF tautomerase via cocrystal structures with phantom hits from virtual screening

    SciTech Connect

    McLean, Larry R.; Zhang, Ying; Li, Hua; Li, Ziyu; Lukasczyk, Ulrike; Choi, Yong-Mi; Han, Zuoning; Prisco, Joy; Fordham, Jeremy; Tsay, Joseph T.; Reiling, Stephan; Vaz, Roy J.; Li, Yi

    2010-10-28

    Biochemical and X-ray crystallographic studies confirmed that hydroxyquinoline derivatives identified by virtual screening were actually covalent inhibitors of the MIF tautomerase. Adducts were formed by N-alkylation of the Pro-1 at the catalytic site with a loss of an amino group of the inhibitor.

  16. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  17. Structure-Activity Relationships and Anti-inflammatory Activities of N-Carbamothioylformamide Analogues as MIF Tautomerase Inhibitors.

    PubMed

    Zhang, Yu; Xu, Lei; Zhang, Zhiqiang; Zhang, Zhiyu; Zheng, Longtai; Li, Dan; Li, Youyong; Liu, Feng; Yu, Kunqian; Hou, Tingjun; Zhen, Xuechu

    2015-09-28

    Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is an attractive therapeutic target for the treatment of inflammatory diseases. In our previous study, 3-[(biphenyl-4-ylcarbonyl)carbamothioyl]amino benzoic acid (compound 1) was discovered as a potent inhibitor of MIF by docking-based virtual screening and bioassays. Here, a series of analogues of compound 1 derived from similarity search and chemical synthesis were evaluated for their MIF tautomerase activities, and their structure-activity relationships were then analyzed. The most potent inhibitor (compound 5) with an IC50 of 370 nM strongly suppressed lipopolysaccharide (LPS)-induced production of TNF-α and IL-6 in a dose-dependent manner and significantly enhanced the survival rate of mice with LPS-induced endotoxic shock from 0 to 35% at 0.5 mg/kg and to 45% at 1 mg/kg, highlighting the therapeutic potential of the MIF tautomerase inhibition in inflammatory diseases.

  18. The vestigial enzyme D-dopachrome tautomerase protects the heart against ischemic injury.

    PubMed

    Qi, Dake; Atsina, Kwame; Qu, Lintao; Hu, Xiaoyue; Wu, Xiaohong; Xu, Bin; Piecychna, Marta; Leng, Lin; Fingerle-Rowson, Günter; Zhang, Jiasheng; Bucala, Richard; Young, Lawrence H

    2014-08-01

    The cellular response to stress involves the recruitment and coordination of molecular signaling pathways that prevent cell death. D-dopachrome tautomerase (DDT) is an enzyme that lacks physiologic substrates in mammalian cells, but shares partial sequence and structural homology with macrophage migration inhibitory factor (MIF). Here, we observed that DDT is highly expressed in murine cardiomyocytes and secreted by the heart after ischemic stress. Antibody-dependent neutralization of secreted DDT exacerbated both ischemia-induced cardiac contractile dysfunction and necrosis. We generated cardiomyocyte-specific DDT knockout mice (Myh6-Cre Ddtfl/fl), which demonstrated normal baseline cardiac size and function, but had an impaired physiologic response to ischemia-reperfusion. Hearts from Myh6-Cre Ddtfl/fl mice exhibited more necrosis and LV contractile dysfunction than control hearts after coronary artery ligation and reperfusion. Furthermore, treatment with DDT protected isolated hearts against injury and contractile dysfunction after ischemia-reperfusion. The protective effect of DDT required activation of the metabolic stress enzyme AMP-activated protein kinase (AMPK), which was mediated by a CD74/CaMKK2-dependent mechanism. Together, our data indicate that cardiomyocyte secretion of DDT has important autocrine/paracrine effects during ischemia-reperfusion that protect the heart against injury.

  19. Initial description of primate-specific cystine-knot Prometheus genes and differential gene expansions of D-dopachrome tautomerase genes

    PubMed Central

    Premzl, Marko

    2015-01-01

    Using eutherian comparative genomic analysis protocol and public genomic sequence data sets, the present work attempted to update and revise two gene data sets. The most comprehensive third party annotation gene data sets of eutherian adenohypophysis cystine-knot genes (128 complete coding sequences), and d-dopachrome tautomerases and macrophage migration inhibitory factor genes (30 complete coding sequences) were annotated. For example, the present study first described primate-specific cystine-knot Prometheus genes, as well as differential gene expansions of D-dopachrome tautomerase genes. Furthermore, new frameworks of future experiments of two eutherian gene data sets were proposed. PMID:25941635

  20. Initial description of primate-specific cystine-knot Prometheus genes and differential gene expansions of D-dopachrome tautomerase genes.

    PubMed

    Premzl, Marko

    2015-06-01

    Using eutherian comparative genomic analysis protocol and public genomic sequence data sets, the present work attempted to update and revise two gene data sets. The most comprehensive third party annotation gene data sets of eutherian adenohypophysis cystine-knot genes (128 complete coding sequences), and d-dopachrome tautomerases and macrophage migration inhibitory factor genes (30 complete coding sequences) were annotated. For example, the present study first described primate-specific cystine-knot Prometheus genes, as well as differential gene expansions of D-dopachrome tautomerase genes. Furthermore, new frameworks of future experiments of two eutherian gene data sets were proposed.

  1. Cooperative Regulation of NSCLC Angiogenic Potential by Macrophage Migration Inhibitory Factor and its Homolog, D-Dopachrome Tautomerase1

    PubMed Central

    Coleman, Arlixer M.; Rendon, Beatriz E.; Zhao, Ming; Qian, Ming-Wei; Bucala, Richard; Xin, Dan; Mitchell, Robert A.

    2009-01-01

    Tumor-derived growth factors and cytokines stimulate neoangiogenesis from surrounding capillaries in order to support tumor growth. Recent studies reveal that macrophage migration inhibitory factor (MIF) expression is increased in lung cancer, particularly non-small cell lung carcinomas (NSCLC). Because MIF has important autocrine effects on normal and transformed cells, we investigated whether autocrine MIF and its only known family member, D-dopachrome tautomerase (D-DT), promote the expression of pro-angiogenic factors CXCL8 and VEGF in NSCLC cells. Our results demonstrate that the expression of CXCL8 and VEGF are strongly reliant upon both the individual and cooperative activities of the two family members. CXCL8 transcriptional regulation by MIF and D-DT appears to involve a signaling pathway that includes the activation of c-Jun-N-terminal Kinase (JNK), c-jun phosphorylation and subsequent AP-1 transcription factor activity. Importantly, human umbilical vein endothelial cell (HUVEC) migration and tube formation induced by supernatants from lung adenocarcinoma cells lacking either or both MIF and D-DT are substantially reduced when compared to normal supernatants. Finally, we demonstrate that the cognate MIF receptor, CD74, is necessary for both MIF and D-DT-induced JNK activation and CXCL8 expression, suggesting its potential involvement in angiogenic growth factor expression. This is the first demonstration of a biological role for D-DT and its synergism with MIF suggests that the combined therapeutic targeting of both family members may enhance current anti-MIF based therapies. PMID:18684922

  2. Plasma Levels of Macrophage Migration Inhibitory Factor and d-Dopachrome Tautomerase Show a Highly Specific Profile in Early Life

    PubMed Central

    Roger, Thierry; Schlapbach, Luregn J.; Schneider, Anina; Weier, Manuela; Wellmann, Sven; Marquis, Patrick; Vermijlen, David; Sweep, Fred C. G. J.; Leng, Lin; Bucala, Richard; Calandra, Thierry; Giannoni, Eric

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic, constitutively expressed, pro-inflammatory cytokine and an important regulator of immune responses. d-dopachrome tautomerase (DDT), a newly described member of the MIF protein superfamily, shares sequence homology and biological activities with MIF. We recently reported that high expression levels of MIF sustain innate immune responses in newborns. Here, we elected to further characterize age-dependent MIF expression and to define whether DDT shares a similar expression profile with MIF. Therefore, we delineated the circulating concentrations of MIF and DDT throughout life using a large cohort of 307 subjects including fetuses, newborns, infants, children, and adults. Compared to levels measured in healthy adults (median: 5.7 ng/ml for MIF and 16.8 ng/ml for DDT), MIF and DDT plasma concentrations were higher in fetuses (median: 48.9 and 29.6 ng/ml), increased further at birth (median: 82.6 and 52.0 ng/ml), reached strikingly elevated levels on postnatal day 4 (median: 109.5 and 121.6 ng/ml), and decreased to adult levels during the first months of life. A strong correlation was observed between MIF and DDT concentrations in all age groups (R = 0.91, P < 0.0001). MIF and DDT levels correlated with concentrations of vascular endothelial growth factor, a protein upregulated under low oxygen tension and implicated in vascular and lung development (R = 0.70, P < 0.0001 for MIF and R = 0.65, P < 0.0001 for DDT). In very preterm infants, lower levels of MIF and DDT on postnatal day 6 were associated with an increased risk of developing bronchopulmonary dysplasia and late-onset neonatal sepsis. Thus, MIF and DDT plasma levels show a highly specific developmental profile in early life, supporting an important role for these cytokines during the neonatal period. PMID:28179905

  3. Effect of MC1R variant allele status on MSH-ligand induction of dopachrome tautomerase in melanocytes co-cultured with keratinocytes.

    PubMed

    Ainger, Stephen A; Wong, Shu S; Roberts, Donald W; Leonard, J Helen; Sturm, Richard A

    2011-08-01

    A co-culture system of melanocytic cells and keratinocytes was used to examine dendricity and dopachrome tautomerase (DCT) responses in low penetrant 'r' homozygote and 'R/+' heterozygote MC1R variant allele expressing cells compared to that of wild-type (WT) cells. The V60L-/- homozygote r variant cells showed similar responses to ligand as WT MC1R strains, while V92M-/- homozygote r variant cells were generally shown to have greater dendricity and express higher DCT than the WT cells, even at basal levels. The R151C+/- heterozygote cells showed similar responses to WT cells, while the R160W+/- and D294H+/- variant cells were reduced in their responses to NDP-MSH, but still had an active cAMP response with forskolin treatment. These responses are consistent with the dominant negative effect of these alleles on the MC1R WT allele that has previously been demonstrated genetically and biochemically.

  4. Macrophage migration inhibitory factor (MIF) family in arthropods: Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain.

    PubMed

    Huang, Wen-Shu; Duan, Li-Peng; Huang, Bei; Wang, Ke-Jian; Zhang, Cai-Liang; Jia, Qin-Qin; Nie, Pin; Wang, Tiehui

    2016-03-01

    The macrophage migration inhibitory factor (MIF) family, consisting of MIF and D-dopachrome tautomerase (DDT) in vertebrates, is evolutionarily ancient and has been found across Kingdoms including vertebrates, invertebrates, plants and bacteria. The mammalian MIF family are chemokines at the top of the inflammatory cascade in combating infections. They also possess enzymatic activities, e.g. DDT catalysis results in the production of 5,6-dihydroxyindole (DHI), a precursor of eumelanin. MIF-like genes are widely distributed, but DDT-like genes have only been described in vertebrates and a nematode. In this report, we cloned a DDT-like gene, for the first time in arthropods, and a second MIF in mud crab. The mud crab MIF family have a three exon/two intron structure as seen in vertebrates. The identification of a DDT-like gene in mud crab and other arthropods suggests that the separation of MIF and DDT preceded the divergence of protostomes and deuterostomes. The MIF family is differentially expressed in tissues of adults and during embryonic development and early life. The high level expression of the MIF family in immune tissues, such as intestine and hepatopancreas, suggests an important role in mud crab innate immunity. Mud crab DDT is highly expressed in early embryos, in megalops and crablets and this coincides with the requirement for melanisation in egg chorion tanning and cuticular hardening in arthropods, suggesting a potential novel role of DDT in melanogenesis via its tautomerase activity to produce DHI in mud crab. The clarification of the presence of both MIF and DDT in this report paves the way for further investigation of their functional roles in immunity and in melanogenesis in mud crab and other arthropods.

  5. Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Sarkar, Souvik; Siddiqui, Asim A; Mazumder, Somnath; De, Rudranil; Saha, Shubhra J; Banerjee, Chinmoy; Iqbal, Mohd S; Adhikari, Susanta; Alam, Athar; Roy, Siddhartha; Bandyopadhyay, Uday

    2015-05-27

    Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 μM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

  6. Structure of the mouse tyrosinase-related protein-2/dopachrome tautomerase (Tyrp2/Dct) gene and sequence of two novel slaty alleles

    SciTech Connect

    Budd, P.S.; Jackson, I.J.

    1995-09-01

    We have isolated the eight exons and 5{prime} and 3{prime} flanking regions of the mouse tyrosinase-related protein-2 (dopachrome tautomerase) gene (Tyrp2/Dct), which is mutated in slaty mice. The gene has a structure that is considerably different from those of other tyrosinase family members in both the number and the position of introns, consistent with the suggestion that the divergence of the family represents an ancient gene duplication. We also identify in the 5{prime} flanking DNA an 11-bp element, the M-box, conserved in other tyrosinase family genes. We have characterized point mutations in two slaty alleles recently identified at the Jackson Laboratory: slaty-2J (slt{sup 2J}) has a similar phenotype to the original slaty (slt) mutation, and slaty light (Slt{sup lt}), which has a more severe effect and is semidominant. We suggest that the slaty-light phenotype is a result of the failure of the enzyme to be correctly targeted to its normal location on the inner face of the melanosomal membrane. 48 refs., 4 figs., 3 tabs.

  7. A new transgenic mouse line for tetracycline inducible transgene expression in mature melanocytes and the melanocyte stem cells using the Dopachrome tautomerase promoter.

    PubMed

    Woods, Susan L; Bishop, J Michael

    2011-04-01

    We have generated a novel transgenic mouse to direct inducible and reversible transgene expression in the melanocytic compartment. The Dopachrome tautomerase (Dct) control sequences we used are active early in the development of melanocytes and so this system was designed to enable the manipulation of transgene expression during development in utero and in the melanocyte stem cells as well as mature melanocytes. We observed inducible lacZ and GFP reporter transgene activity specifically in melanocytes and melanocyte stem cells in mouse skin. This mouse model will be a useful tool for the pigment cell community to investigate the contribution of candidate genes to normal melanocyte and/or melanoma development in vivo. Deregulated expression of the proto-oncogene MYC has been observed in melanoma, however whether MYC is involved in tumorigenesis in pigment cells has yet to be directly investigated in vivo. We have used our system to over-express MYC in the melanocytic compartment and show for the first time that increased MYC expression can indeed promote melanocytic tumor formation.

  8. Activation of MITF by Argan Oil Leads to the Inhibition of the Tyrosinase and Dopachrome Tautomerase Expressions in B16 Murine Melanoma Cells

    PubMed Central

    Villareal, Myra O.; Kume, Sayuri; Bourhim, Thouria; Bakhtaoui, Fatima Zahra; Han, Junkyu; Gadhi, Chemseddoha; Isoda, Hiroko

    2013-01-01

    Argan (Argania spinosa L.) oil has been used for centuries in Morocco as cosmetic oil to maintain a fair complexion and to cure skin pimples and chicken pox pustules scars. Although it is popular, the scientific basis for its effect on the skin has not yet been established. Here, the melanogenesis regulatory effect of argan oil was evaluated using B16 murine melanoma cells. Results of melanin assay using B16 cells treated with different concentrations of argan oil showed a dose-dependent decrease in melanin content. Western blot results showed that the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) proteins were decreased. In addition, there was an increase in the activation of MITF and ERK1/2. Real-time PCR results revealed a downregulation of Tyr, Trp1, Dct, and Mitf mRNA expressions. Argan oil treatment causes MITF phosphorylation which subsequently inhibited the transcription of melanogenic enzymes, TYR and DCT. The inhibitory effect of argan oil on melanin biosynthesis may be attributed to tocopherols as well as the synergistic effect of its components. The results of this study provide the scientific basis for the traditionally established benefits of argan oil and present its therapeutic potential against hyperpigmentation disorders. PMID:23935660

  9. Enzyme kinetics and transport in a system crowded by mobile macromolecules.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2015-11-21

    The dynamics of an elastic network model for the enzyme 4-oxalocrotonate tautomerase is studied in a system crowded by mobile macromolecules, also modeled by elastic networks. The system includes a large number of solvent molecules, as well as substrate and product molecules which undergo catalytic reactions with this hexameric protein. The time evolution of the entire system takes place through a hybrid dynamics that combines molecular dynamics for solute species and multiparticle collision dynamics for the solvent. It is shown that crowding leads to subdiffusive dynamics for the protein, in accord with many studies of diffusion in crowded environments, and increases orientational relaxation times. The enzyme reaction kinetics is also modified by crowding. The effective Michaelis constant decreases with crowding volume fraction, and this decrease is attributed to excluded volume effects, which dominate over effects due to reduced substrate diffusion that would cause the Michaelis constant to increase.

  10. The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.

    PubMed

    Azurmendi, Hugo F; Wang, Susan C; Massiah, Michael A; Poelarends, Gerrit J; Whitman, Christian P; Mildvan, Albert S

    2004-04-13

    trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R

  11. Internal Dynamics and Ionization States of the Macrophage Migration Inhibitory Factor: Comparison Between Wild-Type and Mutant Forms

    SciTech Connect

    Soares, Thereza A.; Lins, Roberto D.; Straatsma, TP; Briggs, J. M.

    2002-11-15

    The macrophage migration inhibitory factor (MIF) is a cytokine which shares a common structural architecture and catalytic strategy with three isomerases: 4-oxalocrotonate tautomerase, 5-carboxymethyl-2-hydroxymuconate isomerase and D-dopachrome tautomerase. A highly conserved N-terminal proline acts as a base\\acid during the proton transfer reaction catalyzed by these enzymes. Such unusual catalytic strategy appears to be possible only due to the N-terminal proline pKa be shifted to 5.0-6.0 units. Mutations of this residue result in a significant decrease of the catalytic activity of MIF. Two hypotheses have been proposed to explain the catalytic inefficiency of MIF: the lower basicity of primary amines with regard to secondary ones and the increased flexibility resulting from the replacement of a proline by residues like glycine. To investigate that, we have performed molecular dynamics simulations of MIF-wt and its mutant P1G as well as calculated the protonation properties of several mutant forms. It has been found that the N-terminal glycine does not show larger fluctuations compared to proline, but the former residue is more exposed to the solvent throughout the simulations. The apparent pKa of these residues displays very little change (as expected from the structural rigidity of MIF) and is not significantly affected by the surrounding ionizable residues. Instead, the hydrophobic character of the active site seems to be the main factor in determining the pKa of the N-terminal residue and the catalytic efficiency of MIF.

  12. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveal Substrate Specificity of Protein Acetyltransferases*

    PubMed Central

    Crosby, Heidi A.; Pelletier, Dale A.; Hurst, Gregory B.; Escalante-Semerena, Jorge C.

    2012-01-01

    N-Lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously. PMID:22416131

  13. [Enzymatic formationof 4-hydroxy-2-oxovalerate using pyrazon-degrading bacteria (author's transl)].

    PubMed

    Blobel, F; Eberspächer, J; Lingens, F

    1976-01-01

    By treatment of 2-hydroxymuconic acid with a partially purified 4-oxalocrotonate decarboxylase 4-hydroxy-2-oxovalerate could be obtained. Both forms of 4-hydroxy-2-oxovalerate, the keto as well as the enol form could be isolated.

  14. Quantum mechanics/molecular mechanics minimum free-energy path for accurate reaction energetics in solution and enzymes: Sequential sampling and optimization on the potential of mean force surface

    NASA Astrophysics Data System (ADS)

    Hu, Hao; Lu, Zhenyu; Parks, Jerry M.; Burger, Steven K.; Yang, Weitao

    2008-01-01

    classical SN2 reaction of Cl-+CH3Cl in solution and the second proton transfer step of the reaction catalyzed by the enzyme 4-oxalocrotonate tautomerase. The activation free energies calculated with this new sequential sampling and optimization approach to the QM/MM-MFEP method agree well with results from other simulation approaches such as the umbrella sampling technique with direct QM/MM dynamics sampling, demonstrating the accuracy of the iterative QM/MM-MFEP method.

  15. Quantum mechanics/molecular mechanics minimum free-energy path for accurate reaction energetics in solution and enzymes: Sequential sampling and optimization on the potential of mean force surface

    SciTech Connect

    Hu Hao; Lu Zhenyu; Parks, Jerry M.; Burger, Steven K.; Yang Weitao

    2008-01-21

    classical S{sub N}2 reaction of Cl{sup -}+CH{sub 3}Cl in solution and the second proton transfer step of the reaction catalyzed by the enzyme 4-oxalocrotonate tautomerase. The activation free energies calculated with this new sequential sampling and optimization approach to the QM/MM-MFEP method agree well with results from other simulation approaches such as the umbrella sampling technique with direct QM/MM dynamics sampling, demonstrating the accuracy of the iterative QM/MM-MFEP method.

  16. Associations of common copy number variants in glutathione S-transferase mu 1 and D-dopachrome tautomerase-like protein genes with risk of schizophrenia in a Japanese population.

    PubMed

    Nakamura, Toru; Ohnuma, Tohru; Hanzawa, Ryo; Takebayashi, Yuto; Takeda, Mayu; Nishimon, Shohei; Sannohe, Takahiro; Katsuta, Narimasa; Higashiyama, Ryoko; Shibata, Nobuto; Arai, Heii

    2015-10-01

    Oxidative-stress, genetic regions of interest (1p13 and 22q11), and common copy number variations (CNVs) may play roles in the pathophysiology of schizophrenia. In the present study, we confirmed associations between schizophrenia and the common CNVs in the glutathione (GSH)-related genes GSTT1, DDTL, and GSTM1 using quantitative real-time polymerase chain reaction analyses of 620 patients with schizophrenia and in 622 controls. No significant differences in GSTT1 copy number distributions were found between patient groups. However, frequencies of characterized CNVs and assumed gain alleles of DDTL and GSTM1 were significantly higher in patients with schizophrenia. In agreement with a previous report, the present data indicate that gains in the CNV alleles DDTL and GSTM1 are genetic risk factors in Japanese patients with schizophrenia, and suggest involvement of micro-inflammation and oxidative stress in the pathophysiology of schizophrenia.

  17. Role of Melanin in Oncogenesis

    DTIC Science & Technology

    2011-02-28

    transient siRNA knockdown of enzymes specifically involved in melanin production- phenylalanine hydroxylase , tyrosinase, tyrosine hydroxylase and...biosynthetic enzymes, tyrosinase (siTYR), tyrosine hydroxylase (TYH), phenlyalanine hydroxylase (siPKU), and dopachrome tautomerase (siDCT). The

  18. Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function.

    PubMed

    Dickerhof, Nina; Schindler, Lisa; Bernhagen, Jürgen; Kettle, Anthony J; Hampton, Mark B

    2015-12-01

    Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.

  19. Irregularities in enzyme assays: The case of macrophage migration inhibitory factor.

    PubMed

    Cisneros, José A; Robertson, Michael J; Valhondo, Margarita; Jorgensen, William L

    2016-06-15

    Inhibitors of human macrophage migration inhibitory factor (MIF) previously reported in the literature have been reexamined by synthesis, assaying for tautomerase activity, and protein crystallography. Substantial inconsistencies between prior and current assay results are noted. They appear to arise from difficulties with the tautomerase substrates, solubility issues, and especially covalent inhibition. Incubation time variation shows that 3, 4, 6, and 9 are covalent or slow-binding inhibitors. Two protein crystal structures are provided; one confirms that the twice-discovered 3 is a covalent inhibitor.

  20. Macrophage migration inhibitory factor (MIF) enzymatic activity and lung cancer.

    PubMed

    Mawhinney, Leona; Armstrong, Michelle E; O' Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2015-04-16

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif (P1G)). Primary tumor growth was significantly attenuated in both Mif-KO and Mif (P1G) mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.

  1. Phenol degradation by Sulfobacillus acidophilus TPY via the meta-pathway.

    PubMed

    Zhou, Wengen; Guo, Wenbin; Zhou, Hongbo; Chen, Xinhua

    2016-09-01

    Due to its toxicity and volatility, phenol must be cleared from the environment. Sulfobacillus acidophilus TPY, which was isolated from a hydrothermal vent in the Pacific Ocean as a moderately thermoacidophilic Gram-positive bacterium, was capable of aerobically degrading phenol. This bacterium could tolerate up to 1300mg/L phenol and degrade 100mg/L phenol in 40h completely at 45°C and pH 1.8 with a maximal degradation rate of 2.32mg/L/h at 38h. Genome-wide search revealed that one gene (TPY_3176) and 14 genes clustered together in two regions with locus tags of TPY_0628-0634 and TPY_0640-0646 was proposed to be involved in phenol degradation via the meta-pathway with both the 4-oxalocrotonate branch and the hydrolytic branch. Real-time PCR analysis of S. acidophilus TPY under phenol cultivation condition confirmed the transcription of proposed genes involved in the phenol degradation meta-pathway. Degradation of 3-methylphenol and 2-methylphenol confirmed that the hydrolytic branch was utilised by S. acidophilus TPY. Phylogenetic analysis revealed that S. acidophilus TPY was closely related to sulphate-reducing bacteria and some Gram-positive phenol-degrading bacteria. This was the first report demonstrating the ability of S. acidophilus to degrade phenol and characterising the putative genes involved in phenol metabolism in S. acidophilus TPY.

  2. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  3. 42 CFR 414.408 - Payment rules.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) Enteral nutrition supplies. (4) OTS orthotics. (h) Rented equipment—(1) Capped rental DME. Subject to the... payment to certain contract suppliers for capped rental DME. (i) Except as specified in paragraph (h)(3... and servicing of rented DME. Separate maintenance and servicing payments are not made for any...

  4. 42 CFR 414.408 - Payment rules.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...) Enteral nutrition supplies. (4) OTS orthotics. (h) Rented equipment—(1) Capped rental DME. Subject to the... payment to certain contract suppliers for capped rental DME. (i) Except as specified in paragraph (h)(3... and servicing of rented DME. Separate maintenance and servicing payments are not made for any...

  5. Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions.

    PubMed

    Johnson, William H; Stack, Tyler M M; Taylor, Stephanie M; Burks, Elizabeth A; Whitman, Christian P

    2016-07-26

    A stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate in meta-fission pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of energy and carbon. The analysis was carried out using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate with the enzymes from Pseudomonas putida mt-2 (Pp) and Leptothrix cholodnii SP-6 (Lc). In both organisms, VPH is in a complex with the preceding enzyme in the pathway, 4-oxalocrotonate decarboxylase (4-OD). In D2O, a deuteron is incorporated stereospecifically at the C-3 and C-5 positions of product by both Pp and Lc enzymes. Accordingly, the complexes generate (3S,5S)-3,5-[di-D]-2-keto-4S-hydroxyhexanoate and (3S,5R)-3,5-[di-D]-2-keto-4R-hydroxy-5-chloropentanoate (4R and 5R due to a priority numbering change). The substitution at C-5 (CH3 or Cl) or the source of the enzyme (Pp or Lc) does not change the stereochemical outcome. One mechanism that can account for the results is the ketonization of the 5-substituted dienol to the α,β-unsaturated ketone (placing a deuteron at C-5 in D2O), followed by the conjugate addition of water (placing a deuteron at C-3). The stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related enzyme, 2-oxo-hept-4-ene-1,7-dioate hydratase, from Escherichia coli C. The combined observations suggest similar mechanisms for these three enzymes that could possibly be common to this group of enzymes.

  6. Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.

    PubMed

    Chauhan, Nikhil; Sharma, Rohit; Hoti, S L

    2015-02-01

    Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plants. Filarial parasites produce two homologues of hMIF viz., MIF-1 and MIF-2, which play important role in the host immune modulation. Earlier, we have characterized MIF-1 (Wba-mif-1) from Wuchereria bancrofti, the major causal organism of human lymphatic filariasis. Here, we are reporting the molecular and biochemical characterization of MIF-2 from this parasite (Wba-mif-2). The complete Wba-mif-2 gene and its cDNA were amplified, cloned and sequenced. The size of Wba-mif-2 gene and cDNA were found to be 4.275 kb and 363 bp, respectively. The gene annotation revealed the presence of a large intron of 3.912 kb interspersed with two exons of 183 bp and 180 bp. The alignment of derived amino acid sequences of Wba-MIF-2 with Wba-MIF-1 showed 44% homology. The conserved CXXC oxido-reductase catalytic site present in Wba-mif-1 was found absent in Wba-mif-2 coding sequence. The amplified Wba-mif-2 cDNA was cloned into an expression vector pRSET-B and transformed into salt inducible Escherichia coli strain GJ1158. The expressed recombinant Wba-MIF-2 protein showed tautomerase activity against L-dopachrome methyl ester and the specific activity was determined to be 18.57±0.77 μmol/mg/min. Three known inhibitors of hMIF tautomerase activity significantly inhibited the tautomerase activity of recombinant Wba-MIF-2. Although the conserved CXXC oxido-reductase motif is absent in Wba-mif-2, the recombinant protein showed significant oxido-reductase activity in the insulin reduction assay, possibly because of the presence of vicinal cysteine residues.

  7. X-Ray Laser Program Final Report for FY92

    DTIC Science & Technology

    1993-07-01

    q..b Ŕ = 4) ot -4-D C’) co E 0 A- 10 .0 0-- (fwz) Ajisuap ~ Igd !jl £ýu 16 large increase in spectral output in this region due to the introduction of...Transfer 34,447 (1985). 26. J. C. Weisheit, J. Quant. Spectrosc. Radiat. Transfer 22,585 (1979). 27. R. C. Mancini , R. F. Joyce, and C. F. Hooper, Jr., J

  8. Structural and Functional Characterization of a Secreted Hookworm Macrophage Migration Inhibitory Factor (MIF) that Interacts with the Human MIF Receptor CD74

    SciTech Connect

    Cho,Y.; Jones, B.; Vermeire, J.; Leng, L.; DiFedele, L.; Harrison, L.; Xiong, H.; Kwong, Y.; Chen, Y.; et al

    2007-01-01

    Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.

  9. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

    PubMed Central

    2016-01-01

    Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  10. Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

    PubMed Central

    Tan, T H; Edgerton, S A; Kumari, R; McAlister, M S; Roe, S M; Nagl, S; Pearl, L H; Selkirk, M E; Bianco, A E; Totty, N F; Engwerda, C; Gray, C A; Meyer, D J; Rowe, S M

    2001-01-01

    cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is

  11. Hazardous Waste Inventory and Disposal Assessment for the Space Shuttle Project. Volume I. Hazardous Waste Inventory.

    DTIC Science & Technology

    1981-03-16

    interim authcrization will be granted by April 1981. With interim authorization, California can operate its own HWP for 2 years after the effective ...became effective on November 19, 1980, require that certain information which was not previously required by California law now be provided on all...8217-4 Ot -j)- C >- 4 z j X - o n L)= : xi ouzo "A,-0Z U Woo 00 o- -J rEW 7S LU ZOS WN O 1>.-C 0 2 X)--O Lu U a: S LW -1,--w -1.11101111W1 OWN -LUI.JO

  12. Microgravity Survey of Wilson Dam Powerplant Switchyards, Florence, Alabama.

    DTIC Science & Technology

    1984-04-01

    ofs -,hsur frce ~O~4 ~ IWLO!4 Ot4CCV 65 IS O9nOLET Fui~ t Ee SECUPI’Y CL.ISFCC CF T.IS -A -,E ’PN Pwas Fr.’. Unclassified j SECURITY CLASSIFICATION OF...35 4 36 20 5.539 4 30 10 5.546 5 40 6 5.533 5 46 20 5.!42 6 78 10 5!27 6 56 9 5 533 7109 rd 5 524 7 66 10 5 !35 8 128 10 5.512 6 78 6 5 521 9 160 40

  13. Origin of adult-type pigment cells forming the asymmetric pigment pattern, in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Yamada, Toshiyuki; Okauchi, Masanori; Araki, Kazuo

    2010-12-01

    The flatfish-specific asymmetric pigment pattern depends on the asymmetric appearance of adult-type pigment cells after the late metamorphic stages. To understand the mechanism enabling the formation of this asymmetric pattern, we investigated the behavior of pigment cell latent precursors in postembryonic Japanese flounder, Paralichthys olivaceus, by analysis of the expression patterns of pigment lineage markers (colony stimulating factor 1 receptor, dopachrome tautomerase, kit) and the DiI (DiO) labeling test for latent precursors. We found that, throughout the larval stages, pigment cell latent precursors were predominantly localized along the dorsal and ventral margins of the flank symmetrically and migrated continuously from these regions to the lateral sides symmetrically, and after late metamorphic stages these precursors differentiated into adult-type pigment cells on the lateral side asymmetrically. We conclude that adult-type pigment cells that form the asymmetric pigment pattern are continuously derived from the dorsal and ventral margins of the flank during larval development.

  14. Stromal-dependent tumor promotion by MIF family members

    PubMed Central

    Mitchell, Robert A.; Yaddanapudi, Kavitha

    2014-01-01

    Solid tumors are composed of a heterogeneous population of cells that interact with each other and with soluble and insoluble factors that, when combined, strongly influence the relative proliferation, differentiation, motility, matrix remodeling, metabolism and microvessel density of malignant lesions. One family of soluble factors that is becoming increasingly associated with pro-tumoral phenotypes within tumor microenvironments is that of the migration inhibitory factor family which includes its namesake, MIF, and its only known family member, D-dopachrome tautomerase (D-DT). This review seeks to highlight our current understanding of the relative contributions of a variety of immune and non-immune tumor stromal cell populations and, within those contexts, will summarize the literature associated with MIF and/or D-DT. PMID:25277536

  15. Synthesis and characterization of melanins from dihydroxyindole-2-carboxylic acid and dihydroxyindole.

    PubMed

    Orlow, S J; Osber, M P; Pawelek, J M

    1992-09-01

    Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 micron filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

    PubMed

    Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian; Rojanasthien, Siripong; Cho, Thomas Yoonsang

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  17. A New Pumpjet Design Theory

    DTIC Science & Technology

    1988-06-30

    THRU K &N* 0103 DOOZOKI,NFR,2 0104 COAK =COA(K) 0105 CUO~T2* COAK +CCK) 0106 DS(K,ISEC) 0Q4*COAK4.0S(K,ISEC) 0107 DT(K,ISEC) -06*COAIC4OT(K,ISEC) 0108 DG...KISEC) 0119 END DO 0120 520 CONTINUE 0121 00540K*2,NFR,2 0122 COAKzCOA(K) 0123 C(K)sT1* COAK +C(K) 0124 0S(K, ISEC)-03- COAK .06(K, ISEC) 0125 OT(K,ISEC...05CAK𔃺T(K,ISEC) 0126 DG(1,K,iSEC)= COAK *RSS .0G(1,K,ISEC) 0127 DH(1,K.ISEC)zCOAK*FCCS+ONC1,K,ISEC) 0128 19 = COAK *RSIA2 0129 17 ZCOAK*FCD 0130 T8 = COAK

  18. Drug repositioning and pharmacophore identification in the discovery of hookworm MIF inhibitors.

    PubMed

    Cho, Yoonsang; Vermeire, Jon J; Merkel, Jane S; Leng, Lin; Du, Xin; Bucala, Richard; Cappello, Michael; Lolis, Elias

    2011-09-23

    The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a "proof-of-concept" for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity.

  19. Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

    PubMed

    Larsen, Hilde A S; Austbø, Lars; Mørkøre, Turid; Thorsen, Jim; Hordvik, Ivar; Fischer, Uwe; Jirillo, Emilio; Rimstad, Espen; Koppang, Erling O

    2012-08-01

    Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

  20. Inhibitory effect of N-adamantyl-3,4-dihydroxybenzamide on melanogenesis in melan-a cells and brown guinea pigs.

    PubMed

    Rho, Ho Sik; Ahn, Soo Mi; Hwang, Jae Sung

    2011-04-01

    To find novel depigmenting agents, a new synthetic compound, N-adamantyl-3,4-dihydroxybenzamide (NADB) was produced and the effects on melanogenesis were investigated. Our results showed that NADB reduced melanin synthesis in a dose-dependent manner in melan-a cells. Tyrosinase activity was also reduced by NADB treatment in melan-a cells. However, NADB did not inhibit tyrosinase activity directly in a cell-free system. Treatment of melan-a cells with NADB caused a marked decrease in protein and mRNA levels of tyrosinase along with tyrosinase-related protein 1 and dopachrome tautomerase. To determine whether NADB reduces skin pigmentation, the dorsal skin of brown guinea pigs was shaved and irradiated with UV for 3 weeks using a solar simulator. Then NADB (2 or 1% in propylene glycol:ethanol:water = 5:3:2) was applied topically twice daily for 4 weeks. Visual inspection and Fontana-Masson staining both demonstrated that NADB resulted in lower skin pigmentation and total epidermal melanin in comparison to vehicle-treated areas. These findings suggest that NADB is useful in the treatment of hyperpigmentation.

  1. Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers

    PubMed Central

    Levin, Mark D.; Lu, Min Min; Petrenko, Nataliya B.; Hawkins, Brian J.; Gupta, Tara H.; Lang, Deborah; Buckley, Peter T.; Jochems, Jeanine; Liu, Fang; Spurney, Christopher F.; Yuan, Li J.; Jacobson, Jason T.; Brown, Christopher B.; Huang, Li; Beermann, Friedrich; Margulies, Kenneth B.; Madesh, Muniswamy; Eberwine, James H.; Epstein, Jonathan A.; Patel, Vickas V.

    2009-01-01

    Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes. Given that dysregulation of intracellular calcium and reactive species has been described in patients with atrial fibrillation, we investigated the role of DCT in this process. Here, we characterize a unique DCT-expressing cell population within murine and human hearts that populated the pulmonary veins, atria, and atrioventricular canal. Expression profiling demonstrated that this population expressed adrenergic and muscarinic receptors and displayed transcriptional profiles distinct from dermal melanocytes. Adult mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias. Cultured primary cardiac melanocyte-like cells were excitable, and those lacking DCT displayed prolonged repolarization with early afterdepolarizations. Furthermore, mice with mutations in the tyrosine kinase receptor Kit lacked cardiac melanocyte-like cells and did not develop atrial arrhythmias in the absence of DCT. These data suggest that dysfunction of melanocyte-like cells in the atrium and pulmonary veins may contribute to atrial arrhythmias. PMID:19855129

  2. Stereochemical Control of Enzymatic Carbon–Carbon Bond‐Forming Michael‐Type Additions by “Substrate Engineering”

    PubMed Central

    Miao, Yufeng; Tepper, Pieter G.; Geertsema, Edzard M.

    2016-01-01

    The enzyme 4‐oxalocrotonate tautomerase (4‐OT) promiscuously catalyzes the Michael‐type addition of acetaldehyde to β‐nitrostyrene derivatives to yield chiral γ‐nitroaldehydes, which are important precursors for pharmaceutically active γ‐aminobutyric acids. In this study, we investigated the effect of different substituents at the aromatic ring of the Michael acceptor on the catalytic efficiency and stereoselectivity of the 4‐OT‐catalyzed acetaldehyde addition reactions. Highly enantioenriched (R)‐ and (S)‐γ‐nitroaldehydes and 4‐substituted chroman‐2‐ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate. Stereochemical control of these enzymatic Michael‐type additions by “substrate engineering” allowed the enantioselective synthesis of valuable γ‐aminobutyric acid precursors. In addition, the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds for preparing biologically active natural products. PMID:27917069

  3. Induction of ambicoloration by exogenous cortisol during metamorphosis of spotted halibut Verasper variegatus.

    PubMed

    Yamada, Toshiyuki; Donai, Hayato; Okauchi, Masanori; Tagawa, Masatomo; Araki, Kazuo

    2011-12-01

    Cortisol, the main glucocorticoid in fish, increases during flatfish metamorphosis and peaks before the surge of thyroxine. A large body of evidence indicates the essential role of thyroxine in flatfish metamorphosis, whereas information on cortisol is limited. We administered cortisol to spotted halibut Verasper variegatus larvae in order to examine the effect on pigmentation during metamorphosis. Administration of 10 μg cortisol per mL of water from before the onset of metamorphosis (stage E) to metamorphic climax (stage G) induced the development of adult type pigment cells on the blind side of the metamorphosed juveniles and increased the occurrence of ambicolored juveniles. When 10 μg/mL cortisol was administered during stage D, stages E-F, stage G or stage H, only the administration during stages E-F induced the development of adult type pigment cells on the blind side. In addition, the expression of the gene dopachrome tautomerase (dct), a marker of melanoblasts, was enhanced at Stage E by cortisol administration. These results clearly indicated, for the first time, the enhancement of pigmentation by exogenous high-dose cortisol. Since endogenous cortisol is secreted in response to various kinds of stress in rearing conditions, these results indicate a possible influence of stress conditions in the occurrence of ambicoloration in flatfish.

  4. Comparative analysis of macrophage migration inhibitory factors (MIFs) from the parasitic nematode Onchocerca volvulus and the free-living nematode Caenorhabditis elegans.

    PubMed

    Ajonina-Ekoti, Irene; Kurosinski, Marc Andre; Younis, Abuelhassan Elshazly; Ndjonka, Dieudonne; Tanyi, Manchang Kingsley; Achukwi, Mbunkah; Eisenbarth, Albert; Ajonina, Caroline; Lüersen, Kai; Breloer, Minka; Brattig, Norbert W; Liebau, Eva

    2013-09-01

    The macrophage migration inhibitory factors (MIFs) from the filarial parasite Onchocerca volvulus (OvMIF) were compared to the MIFs from the free-living nematode Caenorhabditis elegans (CeMIF) with respect to molecular, biochemical and immunological properties. Except for CeMIF-4, all other MIFs demonstrated tautomerase activity. Surprisingly, OvMIF-1 displayed oxidoreductase activity. The strongest immunostaining for OvMIF-1 was observed in the outer cellular covering of the adult worm body, the syncytial hypodermis; moderate immunostaining was observed in the uterine wall. The generation of a strong humoral immune response towards OvMIF-1 and reduced reactivity to OvMIF-2 was indicated by high IgG levels in patients infected with O. volvulus and cows infected with the closely related Onchocerca ochengi, both MIFs revealing identical amino acid sequences. Using Litomosoides sigmodontis-infected mice, a laboratory model for filarial infection, MIFs derived from the tissue-dwelling O. volvulus, the rodent gut-dwelling Strongyloides ratti and from free-living C. elegans were recognized, suggesting that L. sigmodontis MIF-specific IgM and IgG1 were produced during L. sigmodontis infection of mice and cross-reacted with all MIF proteins tested. Thus, MIF apparently functions as a target of B cell response during nematode infection, but in the natural Onchocerca-specific human and bovine infection, the induced antibodies can discriminate between MIFs derived from parasitic or free-living nematodes.

  5. Melanocytes expressing MC1R polymorphisms associated with red hair color have altered MSH-ligand activated pigmentary responses in coculture with keratinocytes.

    PubMed

    Roberts, Donald W; Newton, Richard A; Leonard, J Helen; Sturm, Richard A

    2008-05-01

    The occurrence of red hair and pale skin in individuals, which is associated with UV-radiation sensitivity and increased skin cancer risk, is mainly due to polymorphisms in the melanocortin-1 receptor (MC1R) expressed in melanocytes. We have established a serum free human melanocyte-keratinocyte coculture system to study the behavior and functional abilities of melanocytes expressing MC1R red hair color (RHC) variants in order to identify differences from their wild type (WT) counterparts. This model revealed the importance of elevated calcium levels in promoting strong melanocyte interaction with the surrounding keratinocytes and resulted in a dendritic melanocyte morphology similar to that in skin. However, the dendricity response following agonist activation of the MC1R receptor by NDP-MSH peptide, was markedly enhanced in WT melanocytes in comparison to RHC strains. Analysis of mRNA expression and protein levels of the major pigmentation markers following NDP-MSH treatment distinguished the enzyme dopachrome tautomerase as preferentially upregulated in cocultures of WT strains, with negligible or a much reduced response in melanocytes with RHC variant alleles. These results highlight the use of the coculture system in determining fundamental differences in the physiology of melanocytes expressing RHC MC1R receptors and those of WT genotype, which are likely to contribute to the increased skin cancer risk for individuals that carry these variants.

  6. The Role of DCT in HPV16 Infection of HaCaTs

    PubMed Central

    Aksoy, Pınar; Meneses, Patricio I.

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells. PMID:28095444

  7. Anti-melanogenic effects of resveratryl triglycolate, a novel hybrid compound derived by esterification of resveratrol with glycolic acid.

    PubMed

    Park, Soojin; Seok, Jin Kyung; Kwak, Jun Yup; Choi, Yun-Hyeok; Hong, Seong Su; Suh, Hwa-Jin; Park, Woncheol; Boo, Yong Chool

    2016-07-01

    Resveratrol is known to inhibit cellular melanin synthesis by multiple mechanisms. Glycolic acid (GA) is used in skin care products for its excellent skin penetration. The purpose of this study was to examine the anti-melanogenic effects of resveratryl triglycolate (RTG), a novel hybrid compound of resveratrol and GA, in comparison with resveratrol, GA, resveratryl triacetate (RTA) and arbutin. Resveratrol, RTG, and RTA inhibited the catalytic activity human tyrosinase (TYR) more potently than arbutin or GA did. Their cytotoxic and anti-melanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEMs). The cytotoxicity of RTG was similar to that of resveratrol and RTA. RTG at 3-10 μM decreased melanin levels and cellular TYR activities in α-melanocyte-stimulating hormone-stimulated B16/F10 cells, and L-tyrosine-stimulated HEMs. RTG also suppressed mRNA and protein expression of TYR, tyrosinase-related protein 1, L-3,4-dihydroxyphenylalanine chrome tautomerase, and microphthalmia-associated transcription factor (MITF) in HEMs stimulated with L-tyrosine. This study suggests that, like resveratrol and RTA, RTG can attenuate cellular melanin synthesis effectively through the suppression of MITF-dependent expression of melanogenic enzymes and the inhibition of catalytic activity of TYR enzyme. RTG therefore has potential for use as a cosmeceutical ingredient for skin whitening.

  8. The Role of DCT in HPV16 Infection of HaCaTs.

    PubMed

    Aksoy, Pınar; Meneses, Patricio I

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells.

  9. Drug Repositioning and Pharmacophore Identification in the Discovery of Hookworm MIF Inhibitors

    SciTech Connect

    Y Cho; J Vermeire; J Merkel; L Leng; X Du; R Bucala; M Cappello; E Lolis

    2011-12-31

    The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a 'proof-of-concept' for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity.

  10. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    Bai, Fengwei; Asojo, Oluwatoyin A; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A; Leng, Lin; Koski, Raymond A; Powell, Thomas J; Bucala, Richard; Anthony, Karen G

    2012-08-31

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

  11. Targeting MIF in Cancer: Therapeutic Strategies, Current Developments, and Future Opportunities.

    PubMed

    O'Reilly, Ciaran; Doroudian, Mohammad; Mawhinney, Leona; Donnelly, Seamas C

    2016-05-01

    Strong evidence has been presented linking chronic inflammation to the onset and pathogenesis of cancer. The multifunctional pro-inflammatory protein macrophage migration inhibitory factor (MIF) occupies a central role in the inflammatory pathway and has been implicated in the tumorigenesis, angiogenesis, and metastasis of many cancer phenotypes. This review highlights the current state of the art, which presents MIF, and the second member of the MIF structural superfamily, D-DT (MIF2), as significant mediators in the inflammatory-cancer axis. Although the mechanism by which MIF asserts its biological activity has yet to be fully understood, it has become clear in recent years that for certain phenotypes of cancer, MIF represents a valid therapeutic target. Current research efforts have focused on small molecule approaches that target MIF's unique tautomerase active site and neutralization of MIF with anti-MIF antibodies. These approaches have yielded promising results in a number of preclinical murine cancer models and have helped to increase our understanding of MIF biological activity. More recently, MIF's involvement in a number of key protein-protein interactions, such as with CD74 and HSP90, has been highlighted and provides a novel platform for the development of anti-MIF chemotherapeutic strategies in the future.

  12. Identification of Iguratimod as an Inhibitor of Macrophage Migration Inhibitory Factor (MIF) with Steroid-sparing Potential.

    PubMed

    Bloom, Joshua; Metz, Christine; Nalawade, Saisha; Casabar, Julian; Cheng, Kai Fan; He, Mingzhu; Sherry, Barbara; Coleman, Thomas; Forsthuber, Thomas; Al-Abed, Yousef

    2016-12-16

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis.

  13. MIF: Implications in the Pathoetiology of Systemic Lupus Erythematosus.

    PubMed

    Lang, Tali; Foote, Andrew; Lee, Jacinta P W; Morand, Eric F; Harris, James

    2015-01-01

    Macrophage migration Inhibitory factor (MIF) was one of the earliest pro-inflammatory cytokines to be identified. Increasing interest in this cytokine in recent decades has followed the cloning of human MIF and the generation of Mif(-/-) mice. Deepening understanding of signaling pathways utilized by MIF and putative receptor mechanisms have followed. MIF is distinct from all other cytokines by virtue of its unique induction by and counter regulation of glucocorticoids (GCs). MIF is further differentiated from other cytokines by its structural homology to specific tautomerase and isomerase enzymes and correlative in vitro enzymatic functions. The role of MIF in immune and inflammatory states, including a range of human autoimmune diseases, is now well established, as are the relationships between MIF polymorphisms and a number of inflammatory diseases. Here, we review the known pleiotropic activities of MIF, in addition to novel functions of MIF in processes including autophagy and autophagic cell death. In addition, recent developments in the understanding of the role of MIF in systemic lupus erythematosus (SLE) are reviewed. Finally, we discuss the potential application of anti-MIF strategies to treat human diseases such as SLE, which will require a comprehensive understanding of the unique and complex activities of this ubiquitously expressed cytokine.

  14. 4-(Phenylsulfanyl)butan-2-One Suppresses Melanin Synthesis and Melanosome Maturation In Vitro and In Vivo

    PubMed Central

    Wu, Shing-Yi Sean; Wang, Hui-Min David; Wen, Yi-Shan; Liu, Wangta; Li, Pin-Hui; Chiu, Chien-Chih; Chen, Pei-Chin; Huang, Chiung-Yao; Sheu, Jyh-Horng; Wen, Zhi-Hong

    2015-01-01

    In this study, we screened compounds with skin whitening properties and favorable safety profiles from a series of marine related natural products, which were isolated from Formosan soft coral Cladiella australis. Our results indicated that 4-(phenylsulfanyl)butan-2-one could successfully inhibit pigment generation processes in mushroom tyrosinase platform assay, probably through the suppression of tyrosinase activity to be a non-competitive inhibitor of tyrosinase. In cell-based viability examinations, it demonstrated low cytotoxicity on melanoma cells and other normal human cells. It exhibited stronger inhibitions of melanin production and tyrosinase activity than arbutin or 1-phenyl-2-thiourea (PTU). Also, we discovered that 4-(phenylsulfanyl)butan-2-one reduces the protein expressions of melanin synthesis-related proteins, including the microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (Trp-1), dopachrome tautomerase (DCT, Trp-2), and glycoprotein 100 (GP100). In an in vivo zebrafish model, it presented a remarkable suppression in melanogenesis after 48 h. In summary, our in vitro and in vivo biological assays showed that 4-(phenylsulfanyl)butan-2-one possesses anti-melanogenic properties that are significant in medical cosmetology. PMID:26343635

  15. ISO-66, a novel inhibitor of macrophage migration, shows efficacy in melanoma and colon cancer models.

    PubMed

    Ioannou, Kyriaki; Cheng, Kai Fan; Crichlow, Gregg V; Birmpilis, Anastasios I; Lolis, Elias J; Tsitsilonis, Ourania E; Al-Abed, Yousef

    2014-10-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine, which possesses a contributing role in cancer progression and metastasis and, thus, is now considered a promising anticancer drug target. Many MIF-inactivating strategies have proven successful in delaying cancer growth. Here, we report on the synthesis of ISO-66, a novel, highly stable, small-molecule MIF inhibitor, an analog of ISO-1 with improved characteristics. The MIF:ISO-66 co-crystal structure demonstrated that ISO-66 ligates the tautomerase active site of MIF, which has previously been shown to play an important role in its biological functions. In vitro, ISO-66 enhanced specific and non-specific anticancer immune responses, whereas prolonged administration of ISO-66 in mice with established syngeneic melanoma or colon cancer was non-toxic and resulted in a significant decrease in tumor burden. Subsequent ex vivo analysis of mouse splenocytes revealed that the observed decrease in tumor growth rates was likely mediated by the selective in vivo expansion of antitumor-reactive effector cells induced by ISO-66. Compared to other MIF-inactivating strategies employed in vivo, the anticancer activity of ISO-66 is demonstrated to be of equal or better efficacy. Our findings suggest that targeting MIF, via highly specific and stable compounds, such as ISO-66, may be effective for cancer treatment and stimulation of anticancer immune responses.

  16. Functional characterization of the Plasmodium falciparum and P. berghei homologues of macrophage migration inhibitory factor.

    PubMed

    Augustijn, Kevin D; Kleemann, Robert; Thompson, Joanne; Kooistra, Teake; Crawford, Carina E; Reece, Sarah E; Pain, Arnab; Siebum, Arjan H G; Janse, Chris J; Waters, Andrew P

    2007-03-01

    Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection.

  17. In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells

    PubMed Central

    Allouche, Jennifer; Bellon, Nathalia; Saidani, Manoubia; Stanchina-Chatrousse, Laure; Masson, Yolande; Patwardhan, Anand; Gilles-Marsens, Floriane; Delevoye, Cédric; Domingues, Sophie; Nissan, Xavier; Martinat, Cécile; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

    2015-01-01

    “Café-au-lait” macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases. PMID:26150484

  18. STRUCTURAL CHARACTERISATION OF THE LEISHMANIA MAJOR ORTHOLOGUES OF MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF)

    PubMed Central

    Richardson, Julia M.; Morrison, Lesley S.; Bland, Nicholas D.; Bruce, Sandra; Coombs, Graham H.; Mottram, Jeremy C.; Walkinshaw, Malcolm D.

    2011-01-01

    Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes - highlighting that MIF is not a virulence factor in all species of Leishmania. PMID:19187777

  19. In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells.

    PubMed

    Allouche, Jennifer; Bellon, Nathalia; Saidani, Manoubia; Stanchina-Chatrousse, Laure; Masson, Yolande; Patwardhan, Anand; Gilles-Marsens, Floriane; Delevoye, Cédric; Domingues, Sophie; Nissan, Xavier; Martinat, Cécile; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

    2015-07-21

    "Café-au-lait" macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases.

  20. Polycaprolactone fiber meshes provide a 3D environment suitable for cultivation and differentiation of melanocytes from the outer root sheath of hair follicle.

    PubMed

    Savkovic, Vuk; Flämig, Franziska; Schneider, Marie; Sülflow, Katharina; Loth, Tina; Lohrenz, Andrea; Hacker, Michael Christian; Schulz-Siegmund, Michaela; Simon, Jan-Christoph

    2016-01-01

    Melanocytes differentiated from the stem cells of human hair follicle outer root sheath (ORS) have the potential for developing non-invasive treatments for skin disorders out of a minimal sample: of hair root. With a robust procedure for melanocyte cultivation from the ORS of human hair follicle at hand, this study focused on the identification of a suitable biocompatible, biodegradable carrier as the next step toward their clinical implementation. Polycaprolactone (PCL) is a known biocompatible material used for a number of medical devices. In this study, we have populated electrospun PCL fiber meshes with normal human epidermal melanocytes (NHEM) as well as with hair-follicle-derived human melanocytes from the outer root sheath (HUMORS) and tested their functionality in vitro. PCL fiber meshes evidently provided a niche for melanocytes and supported their melanotic properties. The cells were tested for gene expression of PAX3, PMEL, TYR and MITF, as well as for proliferation, expression of melanocyte marker proteins tyrosinase and glycoprotein 100 (gp100), L-DOPA-tautomerase enzymatic activity and melanin content. Reduced mitochondrial activity and PAX-3 gene expression indicated that the three-dimensional PCL scaffold supported differentiation rather than proliferation of melanocytes. The monitored melanotic features of both the NHEM and HUMORS cultivated on PCL scaffolds significantly exceeded those of two-dimensional adherent cultures.

  1. Reaction of cis-3-chloroacrylic acid dehalogenase with an allene substrate, 2,3-butadienoate: hydration via an enamine.

    PubMed

    Schroeder, Gottfried K; Johnson, William H; Huddleston, Jamison P; Serrano, Hector; Johnson, Kenneth A; Whitman, Christian P

    2012-01-11

    cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH(4) by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The results may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.

  2. Molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass (Dicentrarchus labrax L.).

    PubMed

    Buonocore, Francesco; Randelli, Elisa; Facchiano, Angelo M; Pallavicini, Alberto; Modonut, Martina; Scapigliati, Giuseppe

    2010-08-15

    The macrophage migration inhibitory factor (MIF) is a cytokine produced in numerous cell types, mainly T lymphocytes and macrophages, in response to inflammatory stimuli. In this paper we report the identification of a cDNA encoding a MIF molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and its 3D structure obtained by template-based modelling. The sea bass MIF cDNA consists of 609bp that translates in one reading frame to give the entire molecule containing 115 amino acids. The sequence contains three cysteine residues in conserved positions compared to human MIF and most Teleost fishes, with the exception of zebrafish and carp. The Cys(57)-Ala(58)-Leu(59)-Cys(60) motif, present inside the stretch important for JAB1-interaction and mediator of the thiol-protein oxidoreductase activity of MIF, is conserved in sea bass, together with the Pro(2) residue that is crucial for the tautomerase catalytic activity. Real-time PCR analyses revealed that MIF is constitutively expressed in all selected tissues and organs, with the highest mRNA level observed in thymus. MIF expression was induced after 4h in vitro stimulation of head kidney leukocytes with LPS and decreased after 24h. The predicted 3D model of sea bass MIF has been used to verify the presence of structural requirements for its known biological activities.

  3. Acerola (Malpighia emarginata DC.) Juice Intake Suppresses UVB-Induced Skin Pigmentation in SMP30/GNL Knockout Hairless Mice

    PubMed Central

    Sato, Yasunori; Uchida, Eriko; Aoki, Hitoshi; Hanamura, Takayuki; Nagamine, Kenichi; Kato, Hisanori; Koizumi, Takeshi

    2017-01-01

    Background/Aims Acerola (Malpighia emarginata DC.) is a fruit that is known to contain high amounts of ascorbic acid (AA) and various phytochemicals. We have previously reported that AA deficiency leads to ultraviolet B (UVB)-induced skin pigmentation in senescence marker protein 30 (SMP30)/gluconolactonase (GNL) knockout (KO) hairless mice. The present study was undertaken to investigate the effects of acerola juice (AJ) intake on the skin of UVB-irradiated SMP30/GNL KO mice. Research design/Principal findings Five-week old hairless mice were given drinking water containing physiologically sufficient AA (1.5 g/L) [AA (+)], no AA [AA (-)] or 1.67% acerola juice [AJ]. All mice were exposed to UVB irradiation for 6 weeks. UVB irradiation was performed three times per week. The dorsal skin color and stratum corneum water content were measured every weekly, and finally, the AA contents of the skin was determined. The skin AA and stratum corneum water content was similar between the AA (+) and AJ groups. The L* value of the AA (+) group was significantly decreased by UVB irradiation, whereas AJ intake suppressed the decrease in the L* value throughout the experiment. Moreover, in the AJ group, there was a significant decrease in the expression level of dopachrome tautomerase, an enzyme that is involved in melanin biosynthesis. Conclusion These results indicate that AJ intake is effective in suppressing UVB-induced skin pigmentation by inhibiting melanogenesis-related genes. PMID:28114343

  4. Thrombosis preventive potential of chicory coffee consumption: a clinical study.

    PubMed

    Schumacher, Edit; Vigh, Eva; Molnár, Valéria; Kenyeres, Péter; Fehér, Gergely; Késmárky, Gábor; Tóth, Kálmán; Garai, János

    2011-05-01

    The protective effects of plant polyphenol intake on cardiovascular morbidity and mortality are widely acknowledged. Caffeine-free chicory coffee is a rich source of plant phenolics, including caffeic acid, which inhibits in vitro platelet aggregation, and also phenylpyruvate tautomerase enzymatic activity of the proinflammatory cytokine, macrophage migration inhibitory factor (MIF). To assess whether chicory coffee consumption might confer cardiovascular benefits a clinical intervention study was performed with 27 healthy volunteers, who consumed 300 mL chicory coffee every day for 1 week. The dietary intervention produced variable effects on platelet aggregation, depending on the inducer used for the aggregation test. Whole blood and plasma viscosity were both significantly decreased, along with serum MIF levels, after 1 week of chicory coffee consumption. Moreover, significant improvements were seen in red blood cell deformability. No changes in hematocrit, fibrinogen level or red blood cell counts were detected. The full spectrum of these effects is unlikely to be attributable to a single compound present in chicory coffee, nevertheless, the phenolics, including caffeic acid, are expected to play a substantial role. In conclusion, our study offers an encouraging starting-point to delineate the antithrombotic and antiinflammatory effects of phenolic compounds found in chicory coffee.

  5. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    SciTech Connect

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  6. Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence.

    PubMed

    Ananth, Abhirami A; Tai, Lee-Hwa; Lansdell, Casey; Alkayyal, Almohanad A; Baxter, Katherine E; Angka, Leonard; Zhang, Jiqing; Tanese de Souza, Christiano; Stephenson, Kyle B; Parato, Kelley; Bramson, Jonathan L; Bell, John C; Lichty, Brian D; Auer, Rebecca C

    2016-01-01

    Anti-tumor CD8+ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)-dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8+ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8+ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.

  7. RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

    PubMed

    Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

    2016-01-15

    Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.

  8. The inhibitory effect of glycolic acid and lactic acid on melanin synthesis in melanoma cells.

    PubMed

    Usuki, Akiko; Ohashi, Akiko; Sato, Hirofumi; Ochiai, Yasunobu; Ichihashi, Masamitsu; Funasaka, Yoko

    2003-01-01

    Alpha-hydroxy acids (AHAs) such as glycolic acid (GA) and lactic acid (LA) have been reported to be effective in treating pigmentary lesions such as melasma, solar lentigines, and postinflammatory hyperpigmentation. The mechanism of this effect might be due to epidermal remodeling and accelerated desquamation, which would result in quick pigment dispersion. However, the direct effect of AHAs on melanin synthesis has not yet been well studied. To elucidate such a direct effect of AHAs on melanogenesis, we performed melanin assays, growth curve determinations, Northern and Western blotting for melanogenic proteins [tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2], and tyrosinase and, 4-dihydroxyphenylalaninechrome tautomerase enzyme activity assays using mouse B16 and human melanoma cells. GA or LA (at doses of 300 or 500 microg/ml) inhibited melanin formation in similar dose-dependent manner, without affecting cell growth. Although the mRNA and protein expression or molecular size of tyrosinase, TRP-1 and TRP-2 were not affected, tyrosinase activity was inhibited. To see whether GA and/or LA directly inhibit tyrosinase catalytic function, the effect of GA and LA on human tyrosinase purified from the melanosome-rich large granule fraction of human melanoma cells was performed. GA or LA were shown to inhibit tyrosinase enzyme activity directly, but this effect was not due to the acidity of GA or LA, because adjusting the pH to 5.6 (the pH of GA and LA at concentrations of 2500 microg/ml), did not affect tyrosinase activity. Taken together, these results show that GA and LA suppress melanin formation by directly inhibiting tyrosinase activity, an effect independent of their acidic nature. GA and LA might work on pigmentary lesions not only by accelerating the turnover of the epidermis but also by directly inhibiting melanin formation in melanocytes.

  9. The critical role of macrophage migration inhibitory factor in insulin activity.

    PubMed

    Vujicic, Milica; Senerovic, Lidija; Nikolic, Ivana; Saksida, Tamara; Stosic-Grujicic, Stanislava; Stojanovic, Ivana

    2014-09-01

    Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MIF-KO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIF's tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF antibody (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability.

  10. Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain.

    PubMed

    Li, Xuefeng; Wang, Huijun; Qiu, Pingming; Luo, Hong

    2008-01-01

    It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.

  11. Transcriptome analysis and molecular signature of human retinal pigment epithelium

    PubMed Central

    Strunnikova, N.V.; Maminishkis, A.; Barb, J.J.; Wang, F.; Zhi, C.; Sergeev, Y.; Chen, W.; Edwards, A.O.; Stambolian, D.; Abecasis, G.; Swaroop, A.; Munson, P.J.; Miller, S.S.

    2010-01-01

    Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed ‘signature’ genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases. PMID:20360305

  12. Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.

    PubMed

    El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

    2012-01-01

    Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

  13. MIF family members cooperatively inhibit p53 expression and activity.

    PubMed

    Brock, Stephanie E; Rendon, Beatriz E; Xin, Dan; Yaddanapudi, Kavitha; Mitchell, Robert A

    2014-01-01

    The tumor suppressor p53 is induced by genotoxic stress in both normal and transformed cells and serves to transcriptionally coordinate cell cycle checkpoint control and programmed cell death responses. Macrophage migration inhibitory factor (MIF) is an autocrine and paracrine acting cytokine/growth factor that promotes lung adenocarcinoma cell motility, anchorage-independence and neo-angiogenic potential. Several recent studies indicate that the only known homolog of MIF, D-dopachrome tautomerase (D-DT - also referred to as MIF-2), has functionally redundant activities with MIF and cooperatively promotes MIF-dependent pro-tumorigenic phenotypes. We now report that MIF and D-DT synergistically inhibit steady state p53 phosphorylation, stabilization and transcriptional activity in human lung adenocarcinoma cell lines. The combined loss of MIF and D-DT by siRNA leads to dramatically reduced cell cycle progression, anchorage independence, focus formation and increased programmed cell death when compared to individual loss of MIF or D-DT. Importantly, p53 mutant and p53 null lung adenocarcinoma cell lines were only nominally rescued from the cell growth effects of MIF/D-DT combined deficiency suggesting only a minor role for p53 in these transformed cell growth phenotypes. Finally, increased p53 activation was found to be independent of aberrantly activated AMP-activated protein kinase (AMPK) that occurs in response to MIF/D-DT-deficiency but is dependent on reactive oxygen species (ROS) that mediate aberrant AMPK activation in these cells. Combined, these findings suggest that both p53 wildtype and mutant human lung adenocarcinoma tumors rely on MIF family members for maximal cell growth and survival.

  14. Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.

    PubMed

    Twu, Olivia; Dessí, Daniele; Vu, Anh; Mercer, Frances; Stevens, Grant C; de Miguel, Natalia; Rappelli, Paola; Cocco, Anna Rita; Clubb, Robert T; Fiori, Pier Luigi; Johnson, Patricia J

    2014-06-03

    The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2-associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.

  15. RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

    PubMed Central

    Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

    2016-01-01

    Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

  16. Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform bacterium strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

    PubMed Central

    Guo, Youzhong; Serrano, Hector; Poelarends, Gerrit J.; Johnson, William H.; Hackert, Marvin L.; Whitman, Christian P.

    2013-01-01

    Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal-ion independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide, as well as a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) shares 38% pairwise sequence identity with the Pseudomonas enzyme including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. In order to determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity, but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily. PMID:23781927

  17. Molecular characterization, tissue distribution and functional analysis of macrophage migration inhibitory factor protein (MIF) in Chinese giant salamanders Andrias davidianus.

    PubMed

    Wang, Lixin; Yang, Hui; Li, Fenggang; Zhang, Yingying; Yang, Zhaoxia; Li, Yang; Liu, Xiaolin

    2013-03-01

    The macrophage migration inhibitory factor (MIF) produced in numerous cell types is a multi-functional cytokine mediating both innate and adaptive immune responses. To obtain a better understanding of the innate immune ability of Andrias davidianus and their defensive mechanisms, we identify the A. davidianus MIF (AdMIF) cDNA sequence from a skin cDNA library. The full-length cDNA of AdMIF was of 661bp, consisting of 134bp 5'-terminal UTR, 348bp open reading frame and 179bp 3'-terminal UTR. The deduced protein was composed of 115 amino acids, with an estimated molecular mass of 12.53kDa and a predicted pI of 6.07. AdMIF primary structure is as conserved as the other known sequences. Real time quantitative PCR (qRT-PCR) analysis indicated that AdMIF gene was ubiquitously expressed in selected tissues, with the highest level in liver, moderate level in spleen, intestine, stomach, and the lowest level in heart and skin. The cDNA fragment encoding mature peptide of AdMIF was recombined and expressed in Escherichia coli BL21 (DE3). By means of Ni(2+)-chelating chromatography, the recombinant protein of AdMIF (rAdMIF) was purified successfully. The rAdMIF protein was proved to have enzymatic redox and tautomerase activity in vitro. This study represents the first report for characterization of A. davidianus MIF, demonstrating the successful isolation of MIF from Chinese giant salamanders, and the purified rAdMIF protein is important to produce the monoclonal antibodies and provides a foundation for further investigation of the physiological function of AdMIF.

  18. Coffee enhances the expression of chaperones and antioxidant proteins in rats with nonalcoholic fatty liver disease.

    PubMed

    Salomone, Federico; Li Volti, Giovanni; Vitaglione, Paola; Morisco, Filomena; Fogliano, Vincenzo; Zappalà, Agata; Palmigiano, Angelo; Garozzo, Domenico; Caporaso, Nicola; D'Argenio, Giuseppe; Galvano, Fabio

    2014-06-01

    Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee's beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2'-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver.

  19. Quench behavior of a superconducting accelerator magnet

    SciTech Connect

    McInturff, A D; Sampson, W B; Garber, M; Dahl, P F

    1980-01-01

    Data are presented on the minimum energy required to cause quenches to propagate in an accelerator dipole magnet. The amount of stored energy dissipated into the magnet was measured as a function of dipole excitation current. This in turn determines the maximum coil temperature reached in a given magnet. Quench velocities in the longitudinal direction of the conductor were as high as 11m/sec. The azimuthal velocities or turn to turn velocities were found to be a function of the number of fiberglass layers of insulation that the quench had to cross and were on the order of a few tens of centimeters/sec. The field shape of a given magnet was found to be unchanged for more than 100 quenches. The coil to coil connection and inter-coil splice resistances were found to be less than a namo-ohm and therefore of litle consequence in the cryogenic load considerations. No definitive answers were found on how to decrease the rate of training (130 Gauss/Quench average) required from 4.OT to 5.1T.

  20. A Simple Way to Pattern Mn_12-acetate Thin Films

    NASA Astrophysics Data System (ADS)

    Kim, K.; Seo, D. M.; Means, J.; Viswanathan, M.; Teizer, W.

    2004-03-01

    We have observed that Mn_12-acetate ([Mn_12O_12(CH_3COO)_16(H_2O)_4]ot2CH_3COOHot4H_2O) molecules, dissolved in organic solvents, can be self-assembled along the edge of the Mn_12 solution droplet on a Si/SiO2 substrate as the solvent is evaporated. This phenomenon may be related to the well known "coffee-stain effect"”, which leads to a dense particulate deposit along the edge of a drying droplet of coffee on a solid surface. In our study, we have observed such a deposit of Mn_12-acetate at the perimeter of a droplet, after a dilute solution in various organic solvents has been dried. We investigated how the deposits depend on the evaporation rate. Also, we controlled the concentration of the solution to find its relation to the resulting pattern deposit. By patterning the surface with resist and performing a lift-off we created what are, to our knowledge, the first artificial patterns of Mn_12-acetate. This may allow for convenient thin film devices of Mn_12-acetate and work in this direction is ongoing. This work was supported by the Texas Higher Education Coordinating Board and Texas A University.

  1. The effects of exercise training and caloric restriction on the cardiac oxytocin natriuretic peptide system in the diabetic mouse

    PubMed Central

    Broderick, Tom L; Jankowski, Marek; Gutkowska, Jolanta

    2017-01-01

    Background Regular exercise training (ET) and caloric restriction (CR) are the frontline strategies in the treatment of type 2 diabetes mellitus with the aim at reducing cardiometabolic risk. ET and CR improve body weight and glycemic control, and experimental studies indicate that these paradigms afford cardioprotection. In this study, the effects of combined ET and CR on the cardioprotective oxytocin (OT)–natriuretic peptide (NP) system were determined in the db/db mouse, a model of type 2 diabetes associated with insulin resistance, hyperglycemia, and obesity. Methods Five-week-old male db/db mice were assigned to the following groups: sedentary, ET, and ET + CR. Nonobese heterozygote littermates served as controls. ET was performed on a treadmill at moderate intensity, and CR was induced by reducing food intake by 30% of that consumed by sedentary db/db mice for a period of 8 weeks. Results After 8 weeks, only ET + CR, but not ET, slightly improved body weight compared to sedentary db/db mice. Regardless of the treatment, db/db mice remained hyperglycemic. Hearts from db/db mice demonstrated reduced expression of genes linked to the cardiac OT–NP system. In fact, compared to control mice, mRNA expression of GATA binding protein 4 (GATA4), OT receptor, OT, brain NP, NP receptor type C, and endothelial nitric oxide synthase (eNOS) was decreased in hearts from sedentary db/db mice. Both ET alone and ET + CR increased the mRNA expression of GATA4 compared to sedentary db/db mice. Only ET combined with CR produced increased eNOS mRNA and protein expression. Conclusion Our data indicate that enhancement of eNOS by combined ET and CR may improve coronary endothelial vasodilator dysfunction in type 2 diabetes but did not prevent the downregulation of cardiac expression in the OT–NP system, possibly resulting from the sustained hyperglycemia and obesity in diabetic mice. PMID:28138261

  2. Is the Volume of the Caudate Nuclei Associated With Area of Secondary Hyperalgesia? – Protocol for a 3-Tesla MRI Study of Healthy Volunteers

    PubMed Central

    Asghar, Mohammad Sohail; Wetterslev, Jørn; Pipper, Christian Bressen; Johan Mårtensson, Johan; Becerra, Lino; Christensen, Anders; Nybing, Janus Damm; Havsteen, Inger; Boesen, Mikael; Dahl, Jørgen Berg

    2016-01-01

    (Archived by WebCite at http://www.webcitation.org/6i4OtP0Oi) PMID:27317630

  3. Symmetry breaking, phase separation and anomalous fluctuations in driven granular gas

    NASA Astrophysics Data System (ADS)

    Meerson, Baruch; Pöschel, Thorsten; Sasorov, Pavel V.; Schwager, Thomas

    2003-03-01

    What is the role of noise, caused by the discrete nature of particles, in granular dynamics? We address this question by considering a simple driven granular system: an ensemble of nearly elastically colliding hard spheres in a rectangular box, driven by a rapidly vibrating side wall at zero gravity. The elementary state of this system is a strip of enhanced particle density away from the driving wall. Granular hydrodynamics (GHD) predicts a symmetry breaking instability of this state, when the aspect ratio of the confining box exceeds a threshold value, while the average density of the gas is within a ``spinodal interval". At large aspect ratios this instability leads to phase separation similar to that in van der Waals gas. In the present work (see cond-mat/0208286) we focus on the system behavior around the threshold of the symmetry-breaking instability. We put GHD into a quantitative test by performing extensive event-driven molecular dynamic simulations in 2D. Please watch the movies of the simulations at http://summa.physik.hu-berlin.de/ kies/HD/. We found that the supercritical bifurcation curve, predicted by GHD, agrees with the simulations well below and well above the instability threshold. In a wide region of aspect ratios around the threshold the system is dominated by fluctuations. We checked that the fluctuation strength goes down when the number of particles increases. However, fluctuations remain strong (and the critical region wide) even for as many as 4 ot 10^4 particles. We conclude by suggesting that fluctuations may put a severe limitation on the validity of continuum theories of granular flow in systems with a moderately large number of particles.

  4. Androgens and estradiol-17beta production by porcine uterine cells: In vitro study.

    PubMed

    Franczak, A; Kotwica, G

    2010-01-15

    Porcine (Sus scrofa domestica) uterine slices harvested during both early pregnancy and luteolysis produce steroid hormones. The aim of the present study was to determine (1) which porcine separated uterine cells secrete androgens: androstenedione (A(4)) and testosterone (T), and estradiol-17beta (E(2)) in culture; (2) if the production of A(4), T and E(2) in the uterine cells is regulated by P4 and OT; (3) if uterine tissues expressed cytochrome P450arom gene (CYP19). Uteri were collected on Days 14 to 16 of early pregnancy and the estrous cycle. Enzymatically separated epithelial cells, stromal cells, and myocytes were cultured in vitro for 2, 6, and 12h with control medium, progesterone (P(4); 10(-5) M), oxytocin (OT; 10(-7) M), and both hormones (P(4)+OT). The studied cells secreted A(4), T, and E(2) in vitro. Progesterone served as a substrate for steroid synthesis in the uterine cells. Isolated uterine cells, cultured separately, contributed in equal portion to the basal production of androgens (A(4) and T) during both early pregnancy and luteolysis. In pregnant pigs, the epithelial and stromal cells were rich sources of E(2) compared with myocytes. Myocytes produced E(2) mainly during luteolysis. Pregnant porcine endometrium and myometrium expressed the gene CYP19, which encodes for P450 aromatase, a steroidogenic enzyme. The results indicate an active steroidogenic pathway in porcine uterine cells. The epithelial cells, stromal cells, and myocytes participate in steroid production as an alternative source for their action in pigs.

  5. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined.

  6. Conformational characterization of peptides rich in the cycloaliphatic C alpha,alpha-disubstituted glycine 1-aminocyclononane-1-carboxylic acid.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Valle, G; Toniolo, C; Bonora, G M; Saviano, M; Iacovino, R; Menchise, V; Galdiero, S; Pedone, C; Benedetti, E

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C alpha,alpha-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac9c) and two Ala/Ac9c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives mCIAc-Ac9c-OH and Z-Ac9c-OtBu, the dipeptide pBrBz-(Ac9c)2-OtBu, the tetrapeptide Z-(Ac9c)4-OtBu, and the pentapeptide Z-(Ac9c)5-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac9c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac9c residue is a strong beta-turn and helix former. A comparison with the structural propensity of alpha-aminoisobutyric acid, the prototype of C alpha,alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3-8) is made and the implications for the use of the Ac9c residue in conformationally constrained analogues of bioactive peptides are briefly examined.

  7. Ratio dependence in small number discrimination is affected by the experimental procedure

    PubMed Central

    Agrillo, Christian; Piffer, Laura; Bisazza, Angelo; Butterworth, Brian

    2015-01-01

    Adults, infants and some non-human animals share an approximate number system (ANS) to estimate numerical quantities, and are supposed to share a second, ‘object-tracking,’ system (OTS) that supports the precise representation of a small number of items (up to 3 or 4). In relative numerosity judgments, accuracy depends on the ratio of the two numerosities (Weber’s Law) for numerosities >4 (the typical ANS range), while for numerosities ≤4 (OTS range) there is usually no ratio effect. However, recent studies have found evidence for ratio effects for small numerosities, challenging the idea that the OTS might be involved for small number discrimination. Here we tested the hypothesis that the lack of ratio effect in the numbers 1–4 is largely dependent on the type of stimulus presentation. We investigated relative numerosity judgments in college students using three different procedures: a simultaneous presentation of intermingled and separate groups of dots in separate experiments, and a further experiment with sequential presentation. As predicted, in the large number range, ratio dependence was observed in all tasks. By contrast, in the small number range, ratio insensitivity was found in one task (sequential presentation). In a fourth experiment, we showed that the presence of intermingled distractors elicited a ratio effect, while easily distinguishable distractors did not. As the different ratio sensitivity for small and large numbers has been often interpreted in terms of the activation of the OTS and ANS, our results suggest that numbers 1–4 may be represented by both numerical systems and that the experimental context, such as the presence/absence of task-irrelevant items in the visual field, would determine which system is activated. PMID:26579032

  8. Identification of novel androgen receptor target genes in prostate cancer

    PubMed Central

    Jariwala, Unnati; Prescott, Jennifer; Jia, Li; Barski, Artem; Pregizer, Steve; Cogan, Jon P; Arasheben, Armin; Tilley, Wayne D; Scher, Howard I; Gerald, William L; Buchanan, Grant; Coetzee, Gerhard A; Frenkel, Baruch

    2007-01-01

    Background The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1) – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general

  9. Bacterial Utilization of L-sugars and D-amino Acids

    NASA Technical Reports Server (NTRS)

    Pikuta, Elena; Hoover, Richard B.; Klyce, Brig; Davies, Paul C. W.; Davies, Pauline

    2006-01-01

    The fact that organotrophic organisms on Earth use L-amino acids and D-sugars as an energy source is recognized as one of the universal features of life. The chirality of organic molecules with asymmetric location of group- radicals was described a relatively long time ago. In 1848, Louis Pasteur discovered chiral molecules when he investigated the way that crystals of sodium ammonium paratartrate rotated the plane of polarization of light. He found that the crystal structures represented the underlying asymmetry of molecules that existed in either lea-handed or right-handed forms (enantiomers). Pasteur observed that abiotic (chemical) processes produced mixtures with equal numbers (racemic) of the two forms but that living organisms possessed a molecular asymmetry that included only one of the enantiomers (homochirality). He speculated that the origin of the asymmetry of chiral biomolecules might hold the key to the nature of life. All of the amino acids in proteins (except for Glycine which is symmetrical) exhibit the same absolute steric configuration as L-glyceraldehyde. D-amino acids are never found in proteins, although they do exist in nature and are often found in polypeptide antibiotics. Constitutional sugars of cells, opposite to the amino acids, are the D-enantiomers, and the appearance of L-sugars in Nature is extremely rare. Notwithstanding this fact, the metabolism of some bacteria does have capability to use amino acids and sugars with alternative chirality. This property may be caused by the function of specific enzymes belonging to the class of isomerases (racemases, epimerases, isomerases, tautomerases). In our laboratory, we have investigated several anaerobic bacterial strains, and have found that some of these bacteria are capable of using D-amino acids and L-sugars. Strain BK1 is capable of growth on D-arginine, but its growth characteristics on L-arginine are approximately twice higher. Another alkaliphilic strain SCAT(sup T) (= ATCC BAA-1084

  10. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4.

    PubMed

    Costin, Gertrude-E; Valencia, Julio C; Vieira, Wilfred D; Lamoreux, M Lynn; Hearing, Vincent J

    2003-08-01

    Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosinase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membrane-associated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uw-mutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrichment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ultrastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken together, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the

  11. Bacterial utilization of L-sugars and D-amino acids

    NASA Astrophysics Data System (ADS)

    Pikuta, Elena V.; Hoover, Richard B.; Klyce, Brig; Davies, Paul C. W.; Davies, Pauline

    2006-08-01

    The fact that organotrophic organisms on Earth use L-amino acids and D-sugars as an energy source is recognized as one of the universal features of life. The chirality of organic molecules with asymmetric location of group-radicals was described a relatively long time ago. Louis Pasteur observed that abiotic (chemical) processes produced mixtures with equal numbers (racemic) of the two forms but that living organisms possessed a molecular asymmetry that included only one of the enantiomers (homochirality). He speculated that the origin of the asymmetry of chiral biomolecules might hold the key to the nature of life. All of the amino acids in proteins (except for Glycine which is symmetrical) exhibit the same absolute steric configuration as L-glyceraldehyde. D-amino acids are never found in proteins, although they do exist in nature and are often found in polypeptide antibiotics. Constitutional sugars of cells, opposite to the amino acids, are the D-enantiomers, and the appearance of L-sugars in Nature is extremely rare. Notwithstanding this fact, the metabolism of some bacteria does have the capability to use amino acids and sugars with alternative chirality. This property may be caused by the function of specific enzymes belonging to the class of isomerases (racemases, epimerases, isomerases, tautomerases). In our laboratory, we have investigated several anaerobic bacterial strains, and have found that some of these bacteria are capable of using D-amino acids and L-sugars. Strain BK1 is capable of growth on D-arginine, but its growth characteristics on L-arginine are approximately twice as high. Another alkaliphilic strain SCA T (= ATCC BAA-1084 T = JCM 12857 T = DSM 17722 T = CIP 107910 T) was found to be capable of growth on L-ribose and L-arabinose. It is interesting that this strain was incapable of growth on D-arabinose, which suggests the involvement of some alternative mechanism of enzyme activity. In this paper, we describe the preliminary results of

  12. Liver proteomics in progressive alcoholic steatosis

    SciTech Connect

    Fernando, Harshica; Wiktorowicz, John E.; Soman, Kizhake V.; Kaphalia, Bhupendra S.; Khan, M. Firoze; Shakeel Ansari, G.A.

    2013-02-01

    Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3

  13. Dendritic Cells Take up and Present Antigens from Viable and Apoptotic Polymorphonuclear Leukocytes

    PubMed Central

    Alfaro, Carlos; Suarez, Natalia; Oñate, Carmen; Perez-Gracia, Jose L.; Martinez-Forero, Ivan; Hervas-Stubbs, Sandra; Rodriguez, Inmaculada; Perez, Guiomar; Bolaños, Elixabet; Palazon, Asis; de Sanmamed, Miguel Fernandez; Morales-Kastresana, Aizea; Gonzalez, Alvaro; Melero, Ignacio

    2011-01-01

    Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2d) are coinjected in the footpad of mice with autologous DC (H-2b). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC. PMID:22206007

  14. Oxytocin mitigated the depressive-like behaviors of maternal separation stress through modulating mitochondrial function and neuroinflammation.

    PubMed

    Amini-Khoei, Hossein; Mohammadi-Asl, Ali; Amiri, Shayan; Hosseini, Mir-Jamal; Momeny, Majid; Hassanipour, Mahsa; Rastegar, Mojgan; Haj-Mirzaian, Arya; Mirzaian, Arvin Haj; Sanjarimoghaddam, Hossein; Mehr, Shahram Ejtemaei; Dehpour, Ahmad Reza

    2017-03-01

    Mother-infant contact has a critical role on brain development and behavior. Experiencing early-life adversities (such as maternal separation stress or MS in rodents) results in adaptations of neurotransmission systems, which may subsequently increase the risk of depression symptoms later in life. In this study, we show that Oxytocin (OT) exerted antioxidant and anti-inflammatory properties. Previous studies indicate that neuroinflammation and mitochondrial dysfunction are associated with the pathophysiology of depression. To investigate the antidepressant-like effects of OT, we applied MS paradigm (as a valid animal model of depression) to male mice at postnatal day (PND) 2 to PND 14 (3h daily, 9AM to 12AM) and investigated the depressive-like behaviors of these animals at PND 60 in different groups. Animals in this work were divided into 4 experimental groups: 1) saline-treated, 2) OT-treated, 3) atosiban (OT antagonist)-treated and, 4) OT+ atosiban-treated mice. We used forced swimming test (FST), splash test, sucrose preference test (SPT) and open field test (OFT) for behavioral assessment. Additionally, we used another set of animals to investigate the effects of MS and different treatments on mitochondrial function and the expression of the relevant genes for neuroinflammation. Our results showed that MS provoked depressive- like behaviors in the FST, SPT and splash test. In addition, our molecular findings revealed that MS is capable of inducing abnormal mitochondrial function and immune-inflammatory response in the hippocampus. Further, we observed that treating stressed animals with OT (intracerebroventricular, i.c.v. injection) attenuated the MS-induced depressive-like behaviors through improving mitochondrial function and decreasing the hippocampal expression of immune-inflammatory genes. In conclusion, we showed that MS-induced depressive-like behaviors in adult male mice are associated with abnormal mitochondrial function and immune

  15. Synthesis and characterization of Cu(2)(I,I), Cu(2)(I,II), and Cu(2)(II,II) compounds supported by two phthalazine-based ligands: influence of a hydrophobic pocket.

    PubMed

    Kuzelka, Jane; Mukhopadhyay, Sumitra; Spingler, Bernhard; Lippard, Stephen J

    2004-03-08

    The copper coordination chemistry of two phthalazine-based ligands of differing steric bulk was investigated. A family of dinuclear complexes were prepared from reactions of [Cu(2)(bdptz)(MeCN)(2)](OTf)(2), 1(OTf)(2), where bdptz = 1,4-bis(2,2'-dipyridylmethyl)phthalazine. Treatment of 1(OTf)(2) with NaO(2)CCH(3) afforded the class I mixed-valent compound [Cu(2)(bdptz)(2)](OTf)(3), 2(OTf)(3), by disproportionation of Cu(I). Compound 2(OTf)(3) displays an electron paramagnetic resonance spectrum, with g( parallel ) = 2.25 (A( parallel ) = 169 G) and g( perpendicular ) = 2.06, and exhibits a reversible redox wave at -452 mV versus Cp(2)Fe(+)/Cp(2)Fe. The complex [Cu(2)(bdptz)(micro-OH)(MeCN)(2)](OTf)(3), 3(OTf)(3), was prepared by chemical oxidation of 1 with AgOTf, and exposure of 1 to dioxygen afforded [Cu(2)(bdptz)(micro-OH)(2)](2)(OTs)(4), 4(OTs)(4), which can also be obtained directly from [Cu(H(2)O)(6)](OTs)(2). In compound [Cu(2)(bdptz)(micro-vpy)](OTf)(2), 5(OTf)(2), where vpy = 2-vinylpyridine, the vpy ligand bridges the two Cu(I) centers by using both its pyridine nitrogen and the olefin as donor functionalities. The sterically hindered compounds [Cu(2)(Ph(4)bdptz)(MeCN)(2)](OTf)(2), 6(OTf)(2), and [Cu(2)(Ph(4)bdptz)(micro-O(2)CCH(3))](OTf), 7(OTf), were also synthesized, where Ph(4)bdptz = 1,4-bis[bis(6-phenyl-2-pyridyl)methyl]phthalazine. Complexes 1-7 were characterized structurally by X-ray crystallography. In 6 and 7, the four phenyl rings form a hydrophobic pocket that houses the acetonitrile and acetate ligands. Complex 6 displays two reversible redox waves with E(1/2) values of +41 and +516 mV versus Cp(2)Fe(+)/Cp(2)Fe. Analysis of oxygenated solutions of 6 by electrospray ionization mass spectrometry reveals probable aromatic hydroxylation of the Ph(4)bdptz ligand. The different chemical and electrochemical behavior of 1 versus 6 highlights the influence of a hydrophobic binding pocket on the stability and reactivity of the dicopper(I) centers.

  16. Modeling of both shared and distinct interactions between MIF and its homologue D-DT with their common receptor CD74.

    PubMed

    Meza-Romero, Roberto; Benedek, Gil; Jordan, Kelley; Leng, Lin; Pantouris, Georgios; Lolis, Elias; Bucala, Richard; Vandenbark, Arthur A

    2016-12-01

    D-dopachrome tautomerase (D-DT) shares amino acid sequence similarity, structural architecture and biological activity with the cytokine MIF. Recent studies show that the two protein homologs also bind to the same cell surface receptor, CD74, to activate the ERK1/2 pathway that ultimately leads to pro-inflammatory and pro-survival gene expression. We recently showed that RTL1000 and DRa1-MOG-35-55, two biological drugs with potent anti-inflammatory properties that treat experimental autoimmune encephalomyelitis (EAE) in mice, bind to the cell surface receptor CD74 with high affinity and compete with MIF for binding to the same regions of CD74. Computational modeling of MIF and RTL1000 binding interactions with CD74 predicted the presence of three CD74 binding regions for each MIF homotrimer. Through a similar approach we have now expanded our work to study the D-DT (MIF-2) interaction with CD74 that is mainly defined by three elements scattered throughout the disordered regions of the interacting molecules. The model predicted: (a) a hydrophobic cradle between CD74 and D-DT consisting of N-terminal tyrosine residues of three CD74 monomers arranged in a planar alignment interacts with aromatic amino acid residues located in the disordered D-DT C-terminus; (b) a triad consisting of the E103 residue on one D-DT monomer in close contact with R179 and S181 on one chain of the CD74 trimer forms an intermolecular salt bridge; and (c) amino acid residues on the C-terminus random coil of CD74 chain C form a long interacting area of ∼500Å(2) with a disordered region of D-DT chain B. These three binding elements were also present in MIF/CD74 binding interactions, with involvement of identical or highly similar amino acid residues in each MIF homotrimer that partner with the exact same residues in CD74. Topologically, however, the location of the three CD74 binding regions of the D-DT homotrimer differs substantially from that of the three MIF binding regions. This key

  17. Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions.

    PubMed

    Pavlová, Silvia; Klucska, Karolina; Vašíček, Dušan; Kotwica, Ján; Sirotkin, Alexander V

    2011-10-01

    (2α), but not P(4)) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P(4), OT, PGE(2) and suppression of PGF(2α) release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.

  18. Relationship between concentrations of progesterone, oxytocin, noradrenaline, gene expression and protein level for their receptors in corpus luteum during estrous cycle in the cow.

    PubMed

    Rekawiecki, R; Nowocin, A; Kotwica, J

    2010-06-01

    The aim of these studies was to evaluate of the relationship between luteal concentrations of oxytocin (OT), noradrenaline (NA), progesterone (P4), oxytocin receptors (OT-R) and beta(2)-adrenoreceptors (beta(2)-R) gene expression and their protein level throughout the estrous cycle in cattle. Corpora lutea (CL) collected during days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle were used in these studies. Concentrations of P4, OT and NA were determined in tissue extracts. Gene expression and protein level for OT-R and beta(2)-R were investigated by RT-PCR and Western blot, respectively. Luteal concentration of P4 was higher (P<0.01) on days 6-10 and 11-16 than during days 1-5 and 17-20 of the estrous cycle. Concentration of OT was the highest on days 1-5 and 6-10 of the estrous cycle. This concentration decreased (P<0.01) during days 11-16 reaching the lowest level (P<0.001) on days 17-20 as opposed to days 1-10 of the estrous cycle. Expression of OT-R mRNA was lower on days 6-16 (P<0.05) followed by its increase on days 17-20 as opposed to the expression observed on days 1-5. Expression of OT-R mRNA was negatively correlated (P<0.05) with the profile of OT-R protein level. This latter parameter was the lowest on days 17-20 and 1-5, and the highest on days 6-10 and 11-16. Oxytocin concentration was negatively correlated (P<0.05) with expression of OT-R mRNA and positively correlated (P<0.001) with OT-R protein level. This protein level was only correlated with P4 (r=0.59; P<0.05). Concentration of OT was positively correlated with level of P4 (P<0.001). Concentration of NA was the highest on days 1-5 of the estrous cycle, whereas it was similar or lower (P<0.05) on days 6-21. Expression of mRNA for beta(2)-R was the lowest on days 1-5 and was highly increased (P<0.05) on days 6-16. The expression was the highest (P<0.001) on days 17-21 compared to all others group of the estrous cycle. Protein level for beta(2)-R was the highest on days 1-5 and decreased (P<0

  19. A library of near-infrared integral field spectra of young M-L dwarfs

    NASA Astrophysics Data System (ADS)

    Bonnefoy, M.; Chauvin, G.; Lagrange, A.-M.; Rojo, P.; Allard, F.; Pinte, C.; Dumas, C.; Homeier, D.

    2014-02-01

    Context. At young ages, low surface gravity affects the atmospheric properties of ultracool dwarfs. The impact on medium-resolution near-infrared (NIR) spectra has only been slightly investigated at the M-L transition so far. Aims: We present a library of NIR (1.1-2.45 μm) medium-resolution (R ~ 1500-2000) integral field spectra of 15 young M6-L0 dwarfs. We aim at deriving updated NIR spectral type, luminosity, and physical parameters (Teff, log g, M, L/L⊙) for each source. This work also aims at testing the latest generation of BT-SETTL atmospheric models. Methods: We estimated spectral types using spectral indices and the spectra of young objects classified in the optical. We used the 2010 and 2012 releases of the BT-SETTL synthetic spectral grid and cross-checked the results with the DRIFT-PHOENIX models to derive the atmospheric properties of the sources. Results: We do not find significant differences between the spectra of young companions and those of young isolated brown dwarfs in the same spectral type range. We derive infrared spectral types L0 ± 1, L0 ± 1, M9.5 ± 0.5, M9.5 ± 0.5, M9.25 ± 0.25, M8+0.5-0.75, and M8.5 ± 0.5 for AB Pic b, Cha J110913-773444, USco CTIO 108B, GSC 08047-00232 B, DH Tau B, CT Cha b, and HR7329B, respectively. The BT-SETTL and DRIFT-PHOENIX models yield close Teff and log g estimates for each source. The models seem to show a 600+600-300 K drop in the effective temperature at the M-L transition. Assuming the former temperatures are correct, we then derive new mass estimates that confirm that DH Tau B, USco CTIO 108B, AB Pic b, KPNO Tau 4, OTS 44, and Cha1109 lie inside or at the boundary of the planetary mass range. We combine the empirical luminosities of the M9.5-L0 sources to the Teff to derive semi-empirical radii estimates that do not match "hot-start" evolutionary models predictions at 1-3 Myr. We use complementary data to demonstrate that atmospheric models are able to reproduce the combined optical and infrared