WorldWideScience

Sample records for acireductone dioxygenase isoforms

  1. Characterization of metal binding in the active sites of acireductone dioxygenase isoforms from Klebsiella ATCC 8724.

    Science.gov (United States)

    Chai, Sergio C; Ju, Tingting; Dang, Marina; Goldsmith, Rachel Beaulieu; Maroney, Michael J; Pochapsky, Thomas C

    2008-02-26

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but

  2. Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 (MTCBP-1) acts as an eukaryotic aci-reductone dioxygenase (ARD) in the methionine salvage pathway.

    Science.gov (United States)

    Hirano, Wakako; Gotoh, Isamu; Uekita, Takamasa; Seiki, Motoharu

    2005-06-01

    MTCBP-1 was identified as a protein that binds the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14). Since MTCBP-1 has a putative beta-barrel structure, it is presumably a member of the recently proposed cupin superfamily that contains tremendously diverged functions of proteins in spite of their well-conserved beta-barrel structure. MTCBP-1 shows significant homology to the bacterial aci-reductone dioxygenase (ARD) in the cupin family, which is an enzyme in the methionine salvage pathway (MTA cycle). Since it is difficult to speculate the functions of cupin proteins simply based on their sequence homology, we examined whether the eukaryotic ARD homologs surely function in the methionine metabolism. Under sulfur-depleted conditions, yeast could grow when substrate of MTA cycle was provided. Disruption of the yeast ARD homolog, YMR009w gene, abolished ability of the cells to grow in this culture condition. Re-expression of either the YMR009w or MTCBP-1 gene restored the cell growth. Mutation analysis revealed that the glutamic acid residue in the beta-barrel fold and the N-terminal extension from the beta-barrel fold were found to be important for the activity to restore the growth. Thus, MTCBP-1 isolated as a binding protein for MT1-MMP was demonstrated to function as an ARD-like enzyme in the MTA cycle in yeast.

  3. Acireductone dioxygenase- (ARD-) type reactivity of a nickel(II) complex having monoanionic coordination of a model substrate: product identification and comparisons to unreactive analogues.

    Science.gov (United States)

    Szajna-Fuller, Ewa; Rudzka, Katarzyna; Arif, Atta M; Berreau, Lisa M

    2007-07-09

    A mononuclear Ni(II) complex ([(6-Ph2TPA)Ni(PhC(O)C(OH)C(O)Ph)]ClO4 (1)), supported by the 6-Ph2TPA chelate ligand (6-Ph2TPA = N,N-bis((6-phenyl-2-pyridyl)methyl)-N-((2-pyridyl)methyl)amine) and containing a cis-beta-keto-enolate ligand having a C2 hydroxyl substituent, undergoes reaction with O2 to produce a Ni(II) monobenzoate complex ([(6-Ph2TPA)Ni(O2CPh)]ClO4 (3)), CO, benzil (PhC(O)C(O)Ph), benzoic acid, and other minor unidentified phenyl-containing products. Complex 3 has been identified through independent synthesis and was characterized by X-ray crystallography, 1H NMR, FAB-MS, FTIR, and elemental analysis. A series of cis-beta-keto-enolate Ni(II) complexes supported by the 6-Ph2TPA ligand ([(6-Ph2TPA)Ni(PhC(O)CHC(O)Ph)]ClO4 (4), [(6-Ph2TPA)Ni(CH3C(O)CHC(O)CH3)]ClO4 (5), and [(6-Ph2TPA)Ni(PhC(O)CHC(O)C(O)Ph) (6)) have been prepared and characterized. While these complexes exhibit structural and/or spectroscopic similarity to 1, all are unreactive with O2. The results of this study are discussed in terms of relevance to Ni(II)-containing acireductone dioxygenase enzymes, as well as in the context of recently reported cofactor-free, quercetin, and beta-diketone dioxygenases.

  4. Detection of meta- and ortho-cleavage dioxygenases in bacterial ...

    African Journals Online (AJOL)

    DR. MIKE HORSFALL

    The specific activities of the phenol-degrading enzymes phenol hydroxylase, catechol-1,2-dioxygenase ... reaction catalyzed by 2,3-dioxygenase the meta ... was defined as the initial rate of indigo formation or ... The enzyme reaction was.

  5. Indoleamine 2,3-dioxygenase vaccination

    DEFF Research Database (Denmark)

    Andersen, Mads Hald; Svane, Inge Marie

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. Remarkably, we discovered IDO-specific T cells that can influence adaptive immune reactions in patients with cancer. Further, a recent phase I clinical trial demonstrated long-lasting disease stabilization without toxicity in patien...... with non-small-cell lung cancer (NSCLC) who were vaccinated with an IDO-derived HLA-A2-restricted epitope....

  6. Autoxidation-product-initiated dioxygenases: vanadium-based, record catalytic lifetime catechol dioxygenase catalysis.

    Science.gov (United States)

    Yin, Cindy-Xing; Sasaki, Yoh; Finke, Richard G

    2005-11-14

    In recent work, it was shown that V-containing polyoxometalates such as (n-Bu4N)7SiW9V3O40 or (n-Bu4N)9P2W15V3O62, as well as eight other V-containing precatalysts tested, evolve to a high activity, long catalytic lifetime (> or = 30,000-100,000 total turnovers) 3,5-di-tert-butylcatechol dioxygenase, in which Pierpont's complex [VO(DBSQ)(DTBC)]2 (where DBSQ is 3,5-di-tert-butylsemiquinone and DTBC is the 3,5-di-tert-butylcatecholate dianion) was identified as a common catalyst or catalyst resting state (Yin, C.-X.; Finke, R. G. Vanadium-Based, Extended Catalytic Lifetime Catechol Dioxygenases: Evidence For a Common Catalyst. J. Am. Chem. Soc. 2005, 127 (25), 9003-9013). Herein, those findings are followed up by studies aimed at answering the following questions about this record catalytic lifetime 3,5-di-tert-butylcatechol dioxygenase catalyst: (i) What is the key to how V leaches from, for example, seemingly robust V-containing polyoxometalate precatalysts? (ii) What is the key to the sigmoidal, apparently autocatalytic kinetics observed? (iii) What can be learned about the underlying reactions that form [VO(DBSQ)(DTBC)]2? (iv) Finally, do the answers to (i-iii) lead to any broader insights or concepts? Key findings from the present work include the fact that the reaction involves a novel, autoxidation-product-induced dioxygenase, that is, one in which the undesired autoxidation of the 3,5-di-tert-butylcatechol substrate to the corresponding benzoquinone and H2O2 turns on the desired dioxygenase catalysis via a V-leaching process which eventually yields Pierpont's complex, [VO(DBSQ)(DTBC)]2. Plausible reactions en route to [VO(DBSQ)(DTBC)]2 consistent with the kinetic data, the role of H2O2, and the relevant literature are provided. The results provide a prototype example of the little observed but likely more general concept of an autoxidation-product-initiated reaction. The results also provide considerable simplification of, and insight into, the previously

  7. Heme-containing dioxygenases involved in tryptophan oxidation.

    Science.gov (United States)

    Millett, Elizabeth S; Efimov, Igor; Basran, Jaswir; Handa, Sandeep; Mowat, Christopher G; Raven, Emma Lloyd

    2012-04-01

    Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.

  8. Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases.

    Science.gov (United States)

    Nogales, Juan; Canales, Angeles; Jiménez-Barbero, Jesús; García, José Luis; Díaz, Eduardo

    2005-10-21

    In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.

  9. Hemoglobin: A Nitric-Oxide Dioxygenase

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    Paul R. Gardner

    2012-01-01

    Full Text Available Members of the hemoglobin superfamily efficiently catalyze nitric-oxide dioxygenation, and when paired with native electron donors, function as NO dioxygenases (NODs. Indeed, the NOD function has emerged as a more common and ancient function than the well-known role in O2 transport-storage. Novel hemoglobins possessing a NOD function continue to be discovered in diverse life forms. Unique hemoglobin structures evolved, in part, for catalysis with different electron donors. The mechanism of NOD catalysis by representative single domain hemoglobins and multidomain flavohemoglobin occurs through a multistep mechanism involving O2 migration to the heme pocket, O2 binding-reduction, NO migration, radical-radical coupling, O-atom rearrangement, nitrate release, and heme iron re-reduction. Unraveling the physiological functions of multiple NODs with varying expression in organisms and the complexity of NO as both a poison and signaling molecule remain grand challenges for the NO field. NOD knockout organisms and cells expressing recombinant NODs are helping to advance our understanding of NO actions in microbial infection, plant senescence, cancer, mitochondrial function, iron metabolism, and tissue O2 homeostasis. NOD inhibitors are being pursued for therapeutic applications as antibiotics and antitumor agents. Transgenic NOD-expressing plants, fish, algae, and microbes are being developed for agriculture, aquaculture, and industry.

  10. Structure and mechanism of mouse cysteine dioxygenase

    Science.gov (United States)

    McCoy, Jason G.; Bailey, Lucas J.; Bitto, Eduard; Bingman, Craig A.; Aceti, David J.; Fox, Brian G.; Phillips, George N.

    2006-01-01

    Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical β-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme. PMID:16492780

  11. Functional analysis of alpha-DOX2, an active alpha-dioxygenase critical for normal development in tomato plants.

    Science.gov (United States)

    Bannenberg, Gerard; Martínez, Marta; Rodríguez, María José; López, Miguel Angel; Ponce de León, Inés; Hamberg, Mats; Castresana, Carmen

    2009-11-01

    Plant alpha-dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the alpha-methylene carbon atom of fatty acids. Previously, alpha-DOX1 has been shown to display alpha-dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second alpha-dioxygenase isoform, alpha-DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Slalpha-DOX2 and Atalpha-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Slalpha-DOX2 and Atalpha-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of alpha-DOX1, did not alter the expression of Slalpha-DOX2 or Atalpha-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Slalpha-DOX2 locus and was chosen for functional studies of alpha-DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of Atalpha-DOX2 and Slalpha-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between alpha-DOX2 from tomato and Arabidopsis. However, deletion of Atalpha-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of alpha-DOX2 in plant physiology is species specific.

  12. Exploring the mechanism of tryptophan 2,3-dioxygenase

    Science.gov (United States)

    Thackray, Sarah J.; Mowat, Christopher G.; Chapman, Stephen K.

    2008-01-01

    The haem proteins TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase) are specific and powerful oxidation catalysts that insert one molecule of dioxygen into L-tryptophan in the first and rate-limiting step in the kynurenine pathway. Recent crystallographic and biochemical analyses of TDO and IDO have greatly aided our understanding of the mechanisms employed by these enzymes in the binding and activation of dioxygen and tryptophan. In the present paper, we briefly discuss the function, structure and possible catalytic mechanism of these enzymes. PMID:19021508

  13. Enzymatic activity of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase produced by Gordonia polyisoprenivorans

    Directory of Open Access Journals (Sweden)

    Andréa Scaramal Silva

    2012-01-01

    Full Text Available This study aimed to evaluate the environmental conditions for enzyme activity of catechol 1,2-dioxygenase (C1,2O and catechol 2,3-dioxygenase (C2,3O produced by Gordonia polyisoprenivorans in cell-free and immobilized extracts. The optimum conditions of pH, temperature, time course and effect of ions for enzyme activity were determined. Peak activity of C1,2O occurred at pH 8.0. The isolate exhibited the highest activity of C2,3O at pH 7.0 and 8.0 for the cell-free extract and immobilized extract, respectively. This isolate exhibited important characteristics such as broad range of pH, temperature and time course for enzyme activity.

  14. Histamine H3-receptor isoforms.

    Science.gov (United States)

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  15. Structure and function of dioxygenases in histone demethylation and DNA/RNA demethylation

    Directory of Open Access Journals (Sweden)

    Cheng Dong

    2014-11-01

    Full Text Available Iron(II and 2-oxoglutarate (2OG-dependent dioxygenases involved in histone and DNA/RNA demethylation convert the cosubstrate 2OG and oxygen to succinate and carbon dioxide, resulting in hydroxylation of the methyl group of the substrates and subsequent demethylation. Recent evidence has shown that these 2OG dioxygenases play vital roles in a variety of biological processes, including transcriptional regulation and gene expression. In this review, the structure and function of these dioxygenases in histone and nucleic acid demethylation will be discussed. Given the important roles of these 2OG dioxygenases, detailed analysis and comparison of the 2OG dioxygenases will guide the design of target-specific small-molecule chemical probes and inhibitors.

  16. INDOLEAMINE 2,3-DIOXYGENASE (IDO AND IMMUNE TOLERANCE

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    Coma-del-Corral MJ

    2013-09-01

    Full Text Available SUMMARY: Indoleamine 2,3-dioxygenase (IDO is an intracellular and extrahepatic enzyme predominantly found in many cells, especially macrophages. Tryptophan degradation generates kynurenine, and this pathway of tryptophan metabolism is an effective mechanism for modulating the immune response. The IDO facilitates immune tolerance and is one of the main actors involved in the inhibition of cell proliferation, including activated T cells. IDO induces production of reactive oxygen species (ROS and nitric oxide (NO radicals. Several pathways involved in the regulation of immune response are regulated by redox mechanisms. Reactive oxygen and nitrogen species (ROS-RNS and other redox active molecules play key roles in immunity.

  17. A novel non-heme iron-containing dioxygenase. Chloridazon-catechol dioxygenase from Phenylobacterium immobilis DSM 1986.

    Science.gov (United States)

    Müller, R; Schmitt, S; Lingens, F

    1982-07-01

    Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double-diffusion tests to consist of a single protein species. No cross-reaction was observed with other meta-cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (epsilon = 310 mM -1 cm -1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (epsilon = 1.14 mM -1 cm -1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the pl of 4.5. Surprisingly the enzyme per se is completely inactive, although it contains one non-dialysable iron atom per submit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non-activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta-cleaving enzymes, this enzyme seems to represent a new type of a non-heme iron dioxygenase. We tentatively propose the name Chloridazon-catechol dioxygenase for this enzyme.

  18. Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus

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    Ohira, Koji

    2010-09-01

    Full Text Available Abstract New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO, whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.

  19. 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors: From Chemical Biology to Agrochemicals.

    Science.gov (United States)

    Ndikuryayo, Ferdinand; Moosavi, Behrooz; Yang, Wen-Chao; Yang, Guang-Fu

    2017-10-04

    The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control. HPPD inhibitors have been investigated for agrochemical interests, and some of them have already been commercialized as herbicides. In this review, we mainly focus on the chemical biology of HPPD, discovery of new potential inhibitors, and strategies for engineering transgenic crops resistant to current HPPD-inhibiting herbicides. The conclusion raises some relevant gaps for future research directions.

  20. Structure and function of dioxygenases in histone demethylation and DNA/RNA demethylation

    National Research Council Canada - National Science Library

    Dong, Cheng; Zhang, Heng; Xu, Chao; Arrowsmith, Cheryl H; Min, Jinrong

    2014-01-01

    Iron(II) and 2-oxoglutarate (2OG)-dependent dioxygenases involved in histone and DNA/RNA demethylation convert the cosubstrate 2OG and oxygen to succinate and carbon dioxide, resulting in hydroxylation of the methyl group...

  1. Chemical components from Aloe and their inhibition of indoleamine 2, 3-dioxygenase

    Directory of Open Access Journals (Sweden)

    Ya Nan Sun

    2017-01-01

    Abbreviation used: IDO: inhibit indoleamine 2, 3-dioxygenase, TMS: tetramethylsilane, HMQC: heteronuclear multiple quantum correlation, HMBC: heteronuclear multiple bond correlation, COSY: 1H-1H correlation spectroscopy, ESI-MS: Electrospray ionization mass spectrometry, DMSO: dimethyl sulfoxide

  2. Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

    OpenAIRE

    Zylstra, G J; Wackett, L P; Gibson, D T

    1989-01-01

    Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was o...

  3. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  4. Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31

    NARCIS (Netherlands)

    Mars, Astrid E.; Kingma, Jaap; Kaschabek, Stefan R.; Reineke, Walter; Janssen, Dick B.

    1999-01-01

    Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth, A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2

  5. Overexpression of Crocus carotenoid cleavage dioxygenase, CsCCD4b, in Arabidopsis imparts tolerance to dehydration, salt and oxidative stresses by modulating ROS machinery.

    Science.gov (United States)

    Baba, Shoib Ahmad; Jain, Deepti; Abbas, Nazia; Ashraf, Nasheeman

    2015-09-15

    Apocarotenoids modulate vital physiological and developmental processes in plants. These molecules are formed by the cleavage of carotenoids, a reaction catalyzed by a family of enzymes called carotenoid cleavage dioxygenases (CCDs). Apocarotenoids like β-ionone and β-cyclocitral have been reported to act as stress signal molecules during high light stress in many plant species. In Crocus sativus, these two apocarotenoids are formed by enzymatic cleavage of β-carotene at 9, 10 and 7, 8 bonds by CsCCD4 enzymes. In the present study three isoforms of CsCCD4 were subjected to molecular modeling and docking analysis to determine their substrate specificity and all the three isoforms displayed high substrate specificity for β-carotene. Further, expression of these three CsCCD4 isoforms investigated in response to various stresses revealed that CsCCD4a and CsCCD4b exhibit enhanced expression in response to dehydration, salt and methylviologen, providing a clue towards their role in mediating plant defense response. This was confirmed by overexpressing CsCCD4b in Arabidopsis. The transgenic plants developed longer roots and possessed higher number of lateral roots. Further, overexpression of CsCCD4b imparted enhanced tolerance to salt, dehydration and oxidative stresses as was evidenced by higher survival rate, increased relative root length and biomass in transgenic plants as compared to wild type. Transgenic plants also displayed higher activity and expression of reactive oxygen species (ROS) metabolizing enzymes. This indicates that β-ionone and β-cyclocitral which are enzymatic products of CsCCD4b may act as stress signals and mediate reprogramming of stress responsive genes which ultimately leads to plant defense.

  6. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  7. Structure of the human 4-hydroxyphenylpyruvic acid dioxygenase gene (HPD)

    Energy Technology Data Exchange (ETDEWEB)

    Awata, H.; Endo, F.; Matsuda, I. [Kumamoto Univ. (Japan)

    1994-10-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and developmentally regulated in mammals, and a genetic deficiency in this enzyme in humans and mice leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human gene libraries. The human HPD gene is over 30 kb long and is split into 14 exons. The extract sizes and boundaries of exon blocks were determined, and all of the splice donor and acceptor sites conformed to the GT/AG rule. Analysis of the 5{prime} flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5{prime} flanking region of the gene are similar to those of other genes that are specifically expressed in hepatocytes and that are developmentally regulated. 41 refs., 2 figs., 1 tab.

  8. Engineering Non-Heme Mono- and Dioxygenases for Biocatalysis

    Directory of Open Access Journals (Sweden)

    Adi Dror

    2012-09-01

    Full Text Available Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

  9. Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M

    Directory of Open Access Journals (Sweden)

    Arezoo Tavakoli

    2016-09-01

    Full Text Available In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp, and the product was cloned into a pGEM-T vector.The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3. The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax and Michaelis constant (Km were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS.

  10. Chloridazon-catechol dioxygenases, a distinct group of meta-cleaving enzymes.

    Science.gov (United States)

    Schmitt, S; Müller, R; Wegst, W; Lingens, F

    1984-02-01

    We previously described a new meta-cleaving enzyme, termed chloridazon-catechol dioxygenase. The present paper describes the comparison of this enzyme with the meta-cleaving enzymes of eighteen strains of soil bacteria isolated with various aromatic compounds. Four of these strains were isolated with the herbicide chloridazon, six with the analgeticum aminopyrine and one with the analgeticum antipyrine as sole carbon source. These strains all belonged to a new type of bacteria, called Phenylobacteria. The seven other strains were isolated with aromatic compounds such as toluene, 3-phenylpropionate, benzoate, papaverine and 4-chlorobenzoate, and belonged to various species including Pseudomonas, Acinetobacter and Nocardia. In double diffusion experiments with antibodies, prepared against chloridazon-catechol dioxygenase, extracts from the eleven strains of Phenylobacteria gave a cross reaction, whereas the extracts of the seven other strains showed no reaction. The enzymes of the eleven positive strains showed the same characteristic kinetic behaviour as the previously described enzyme. In contrast to catechol 2, 3-dioxygenase they needed the addition of exogenous Fe2+ ions for activity. On ion-exchange chromatography they emerged at the same buffer concentration as chloridazon-catechol dioxygenase. In polyacrylamide electrophoresis they migrated identically. The linkage map derived from the activities of the various enzymes with 10 different substrates revealed an identity of more than 80% for these eleven enzymes. So the meta-cleaving enzymes of the Phenylobacteria seem to form a distinct group among the non-heme iron-containing dioxygenases.

  11. Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM.

    Science.gov (United States)

    Booth, Elizabeth S; Basran, Jaswir; Lee, Michael; Handa, Sandeep; Raven, Emma L

    2015-12-25

    The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD(+). The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237-244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-methyl-L-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.

  12. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

  13. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

    Energy Technology Data Exchange (ETDEWEB)

    Mesarch, M.B.; Nakatsu, C.H.; Nies, L.

    2000-02-01

    Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primes that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10{sup 2} to 10{sup 3} gene copies, which was lowered to 10{sup 0} to 10{sup 1} gene copies of hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR and a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

  14. Crystal structure of thermostable catechol 2,3-dioxygenase determined by multiwavelength anomalous dispersion method

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The selenomethionyl derivative of the thermostable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed,purified and crystallized. By using multiwave length anomalous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined.TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1~153 and 153~319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.Kevwords: multiwavelength anomalous dispersion (MAD), X-ray diffraction, thermostable catechol 2,3-dioxygenase, crystal structure,synchrotron light source.

  15. Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis.

    Science.gov (United States)

    Su, C; Brodowsky, I D; Oliw, E H

    1995-01-01

    Linoleic acid is sequentially converted to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1-6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3-0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 microM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence of p-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Spectroscopic and equilibrium studies of ligand and organic substrate binding to indolamine 2,3-dioxygenase.

    Science.gov (United States)

    Sono, M

    1990-02-13

    The binding of a number of ligands to the heme protein indolamine 2,3-dioxygenase has been examined with UV-visible absorption and with natural and magnetic circular dichroism spectroscopy. Relatively large ligands (e.g., norharman) which do not readily form complexes with myoglobin and horseradish peroxidase (HRP) can bind to the dioxygenase. Except for only a few cases (e.g., 4-phenylimidazole) for the ferric dioxygenase, a direct competition for the enzyme rarely occurs between the substrate L-tryptophan (Trp) and the ligands examined. L-Trp and small heme ligands (CN-,N3-,F-) markedly enhance the affinity of each other for the ferric enzyme in a reciprocal manner, exhibiting positive cooperativity. For the ferrous enzyme, L-Trp exerts negative cooperativity with some ligands such as imidazoles, alkyl isocyanides, and CO binding to the enzyme. This likely reflects the proximity of the Trp binding site to the heme iron. Other indolamine substrates also exert similar but smaller cooperative effects on the binding of azide or ethyl isocyanide. The pH dependence of the ligand affinity of the dioxygenase is similar to that of myoglobin rather than that of HRP. These results suggest that indolamine 2,3-dioxygenase has the active-site heme pocket whose environmental structure is similar to, but whose size is considerably larger than, that of myoglobin, a typical O2-binding heme protein. Although the L-Trp affinity of the ferric cyanide and ferrous CO enzyme varies only slightly between pH 5.5 and 9.5, the unligated ferric and ferrous enzymes have considerably higher affinity for L-Trp at alkaline pH than at acidic pH. L-Trp binding to the ferrous dioxygenase is affected by an ionizable residue with a pKa value of 7.3.

  17. Crystal Structures of Fe2+ Dioxygenase Superoxo, Alkylperoxo, and Bound Product Intermediates

    OpenAIRE

    Kovaleva, Elena G.; Lipscomb, John D.

    2007-01-01

    We report the structures of three intermediates in the O2 activation and insertion reactions of an extradiol ring-cleaving dioxygenase. A crystal of Fe2+-containing homoprotocatechuate 2,3-dioxygenase was soaked in the slow substrate 4-nitrocatechol in a low O2 atmosphere. The X-ray crystal structure shows that three different intermediates reside in different subunits of a single homotetrameric enzyme molecule. One of these is the key substrate-alkylperoxo-Fe2+ intermediate, which has been p...

  18. Enzymology of the carotenoid cleavage dioxygenases: reaction mechanisms, inhibition and biochemical roles.

    Science.gov (United States)

    Harrison, Peter J; Bugg, Timothy D H

    2014-02-15

    Carotenoid cleavage dioxygenases (CCDs) are a large family of non-heme iron (II) dependent enzymes. CCDs catalyse the selective oxidative cleavage of carotenoids to produce apocarotenoids. Apocarotenoid derived molecules form important signalling molecules in plants in the form of abscisic acid and strigolactone and in mammals in the form of retinal. Very little is known biochemically about the CCDs and only a handful of CCDs have been biochemically characterised. Mechanistically, debate surrounds whether CCDs utilise a mono or dioxygenase mechanism. Here, we review the biochemical roles of CCDs, discuss the mechanisms by which CCD cleavage is proposed to occur, and discuss recent reports of selective CCD enzyme inhibitors.

  19. Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from Saccharomyces cerevisiae

    OpenAIRE

    Hogan, Deborah A; Auchtung, Thomas A.; Hausinger, Robert P.

    1999-01-01

    The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/α-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/α-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being is...

  20. FSH isoform pattern in classic galactosemia

    OpenAIRE

    Gubbels, C.S.; Thomas, C. M.; Wodzig, K.W.H.; Olthaar, A. J.; Jaeken, J.; Sweep, F.C.; Rubio-Gozalbo, M.E.

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  1. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Natural CD4+ T-cell responses against indoleamine 2,3-dioxygenase

    DEFF Research Database (Denmark)

    Munir, Shamaila; Larsen, Stine Kiaer; Iversen, Trine Zeeberg

    2012-01-01

    The enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tum...

  3. An iron-oxygen intermediate formed during the catalytic cycle of cysteine dioxygenase.

    Science.gov (United States)

    Tchesnokov, E P; Faponle, A S; Davies, C G; Quesne, M G; Turner, R; Fellner, M; Souness, R J; Wilbanks, S M; de Visser, S P; Jameson, G N L

    2016-07-07

    Cysteine dioxygenase is a key enzyme in the breakdown of cysteine, but its mechanism remains controversial. A combination of spectroscopic and computational studies provides the first evidence of a short-lived intermediate in the catalytic cycle. The intermediate decays within 20 ms and has absorption maxima at 500 and 640 nm.

  4. Spontaneous cytotoxic T-Cell reactivity against indoleamine 2,3-dioxygenase-2

    DEFF Research Database (Denmark)

    Sørensen, Rikke Bæk; Køllgaard, Tania; Andersen, Rikke Sick;

    2011-01-01

    Several lines of data have suggested a possible link between the indoleamine 2,3-dioxygenase (IDO)-like protein IDO2 and cancer. First, IDO2 expression has been described in human tumors, including renal, gastric, colon, and pancreatic tumors. Second, the apparent selective inhibition of IDO2...

  5. The S. pombe histone H2A dioxygenase Ofd2 regulates gene expression during hypoxia.

    Directory of Open Access Journals (Sweden)

    David Lando

    Full Text Available Post-translational modification of histone proteins are known to play an important role in regulating chromatin structure. In an effort to find additional histone modifications we set out to screen enzymes of the 2-oxoglutarate and Fe(II-dependent (2-OG-Fe(II dioxygenase family for activity towards histones. Here we show that the Schizosaccharomyces pombe 2-OG-Fe(II dioxygenase domain containing protein-2 (Ofd2 is a histone H2A dioxygenase enzyme. Using a combination of peptide screening and alanine scanning substitution analysis, we identify an HxxLR motif in H2A as a substrate for Ofd2 activity. Transcriptional profiling indicates that Ofd2 regulates the repression of oxidative phosphorylation genes during hypoxic stress. We show that Ofd2 is recruited to the 5' end of oxidative phosphorylation genes specifically during hypoxia and that it uses its dioxygenase activity to regulate their transcription. Together, these data uncover a novel histone H2A modifying activity involved in the regulation of gene expression during hypoxia.

  6. Structures of aminophenol dioxygenase in complex with intermediate, product and inhibitor.

    Science.gov (United States)

    Li, De Feng; Zhang, Jia Yue; Hou, Yan Jie; Liu, Lei; Hu, Yonglin; Liu, Shuang Jiang; Wang, Da Cheng; Liu, Wei

    2013-01-01

    Dioxygen activation by nonhaem Fe(II) enzymes containing the 2-His-1-carboxylate facial triad has been extensively studied in recent years. Here, crystal structures of 2-aminophenol 1,6-dioxygenase, an enzyme that represents a minor group of extradiol dioxygenases and that catalyses the ring opening of 2-aminophenol, in complex with the lactone intermediate (4Z,6Z)-3-iminooxepin-2(3H)-one and the product 2-aminomuconic 6-semialdehyde and in complex with the suicide inhibitor 4-nitrocatechol are reported. The Fe-ligand binding schemes observed in these structures revealed some common geometrical characteristics that are shared by the published structures of extradiol dioxygenases, suggesting that enzymes that catalyse the oxidation of noncatecholic compounds are very likely to utilize a similar strategy for dioxygen activation and the fission of aromatic rings as the canonical mechanism. The Fe-ligation arrangement, however, is strikingly enantiomeric to that of all other 2-His-1-carboxylate enzymes apart from protocatechuate 4,5-dioxygenase. This structural variance leads to the generation of an uncommon O(-)-Fe(2+)-O(-) species prior to O(2) binding, which probably forms the structural basis on which APD distinguishes its specific substrate and inhibitor, which share an analogous molecular structure.

  7. A biological pathway linking inflammation and depression: activation of indoleamine 2,3-dioxygenase

    Directory of Open Access Journals (Sweden)

    Christmas DM

    2011-07-01

    Full Text Available David M Christmas, JP Potokar, Simon JC DaviesAcademic Unit of Psychiatry, School of Social and Community Medicine, University of Bristol, Bristol, UK A presentation relating to this manuscript was made by Dr David Christmas at the 9th International Meeting on Clinical Pharmacology in Psychiatry (9th IMCPP in Copenhagen, Denmark in September 2010Abstract: This article highlights the evidence linking depression to increased inflammatory drive and explores putative mechanisms for the association by reviewing both preclinical and clinical literature. The enzyme indoleamine 2,3-dioxygenase is induced by proinflammatory cytokines and may form a link between immune functioning and altered neurotransmission, which results in depression. Increased indoleamine 2,3-dioxygenase activity may cause both tryptophan depletion and increased neurotoxic metabolites of the kynurenine pathway, two alterations which have been hypothesized to cause depression. The tryptophan-kynurenine pathway is comprehensively described with a focus on the evidence linking metabolite alterations to depression. The use of immune-activated groups at high risk of depression have been used to explore these hypotheses; we focus on the studies involving chronic hepatitis C patients receiving interferon-alpha, an immune activating cytokine. Findings from this work have led to novel strategies for the future development of antidepressants including inhibition of indoleamine 2,3-dioxygenase, moderating the cytokines which activate it, or addressing other targets in the kynurenine pathway.Keywords: depression, inflammation, indoleamine 2,3-dioxygenase, kynurenine, serotonin, tryptophan

  8. 4-Nitrocatechol as a colorimetric probe for non-heme iron dioxygenases.

    Science.gov (United States)

    Tyson, C A

    1975-03-10

    4-Nitrocatechol is examined as an active site probe for non-heme iron dioxygenases and found to be of value, particularly with those containing iron in the Fe(II) oxidation state. 4-Nitrocatechol is astrong competitive inhibitor of substrate oxygenation by protocatechuate 3,4-dioxygenase, forming a reversible complex with this enzyme, and by pyrocatechase. The number of binding sites per enzyme molecule titrated spectrophotometrically with 4-nitrocatechol agrees with results from previous studies with either the principal substrate or other analogues, as expected of an effective probe. Despite these facts and the observation that both enzymes cleave the same substrates at the same carbon-carbon bond, the optical and electron paramagnetic resonance (EPR) spectra of their 4-nitrocatechol complexes are remarkably different. The 4-nitocatechol-protocatechuate 3,4-dioxygenase optical spectra resemble that of the 4-nitrocatecholate ion shifted 20 to 30 nm to longer wavelength. Concomitant with this change the EPR signal centered at g equal 4.28 shows increased rhombicity (g values at 4.74, 4.28, and 3.74). In contrast, the spectrum of the 4-nitrocatechol-pyrocatechase complex has a maximum at the same wavelength as that of a 1:1 solution of free Fe(II) and 4-nitrocatechol in the absence of enzyme after titration of the catecholic protons with base and the g equal 4.28 EPR signal is not resolved at liquid N-2 temperature. These changes are interpreted as resulting in part from a pronounced change in the ligand fields about the irons at the active sites which in the case of protocatechuate 3,4-dioxygenase leads to enzyme inactivation. The results also are the first indication that substrate analogues change their ionization form upon complexation with Fe (III) dioxygenases. The interaction of the probe with metapyrocatechase, an Fe(III) containing dioxygenase, and with several additional oxygenases and hydroperoxidases is also briefly examined. The probe is not specific

  9. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

    Full Text Available Objective. To investigate expression of vascular endothelial growth factor (VEGF antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

  10. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

    Laura; A; Perez-Casellas; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

  11. Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

    Science.gov (United States)

    Hupert-Kocurek, Katarzyna; Guzik, Urszula; Wojcieszyńska, Danuta

    2012-01-01

    This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

  12. Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols.

    Science.gov (United States)

    Morrow, William P; Sardar, Sinjinee; Thapa, Pawan; Hossain, Mohammad S; Foss, Frank W; Pierce, Brad S

    2017-10-01

    Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2(-)). Previous chemical rescue studies identified a putative Fe(III)-O2(-) intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Cloning, expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain.

    Science.gov (United States)

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

    2016-10-02

    The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858 bp encoding a polypeptide of 285 amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2 µM and 0.987 µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.

  14. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  15. p53 isoforms change p53 paradigm

    OpenAIRE

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  16. Isoforms of receptors of fibroblast growth factors.

    Science.gov (United States)

    Gong, Siew-Ging

    2014-12-01

    The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

  17. Absolute quantitation of protein posttranslational modification isoform.

    Science.gov (United States)

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

  18. Discovery of Key Dioxygenases that Diverged the Paraherquonin and Acetoxydehydroaustin Pathways in Penicillium brasilianum.

    Science.gov (United States)

    Matsuda, Yudai; Iwabuchi, Taiki; Fujimoto, Takayuki; Awakawa, Takayoshi; Nakashima, Yu; Mori, Takahiro; Zhang, Huiping; Hayashi, Fumiaki; Abe, Ikuro

    2016-09-28

    Paraherquonin (1), a fungal meroterpenoid produced by Penicillium brasilianum NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of 1 (the prh cluster) and elucidated the pathway up to berkeleydione (2), which serves as the key intermediate for the biosynthesis of 1 as well as many other meroterpenoids. Interestingly, the nonheme iron and α-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford 2 from preaustinoid A1 (6), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, P. brasilianum MG11, which produces acetoxydehydroaustin instead of 1, was found to have a gene cluster nearly identical to the prh cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts 6 but produces preaustinoid A3 (17) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species.

  19. A two-electron shell game: Intermediates of the extradiol-cleaving catechol dioxygenases

    Science.gov (United States)

    Fielding, Andrew J.

    2014-01-01

    Extradiol catechol ring-cleaving dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase (HPCD) are summarized with the objective of showing how Nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active site metals, introducing active site amino acid substituted variants, and using substrates with different electron donating capacities. While each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic and computational analysis of the various intermediates shed light on how catalytic efficiency can be achieved. PMID:24615282

  20. Indoleamine 2,3-dioxygenases with very low catalytic activity are well conserved across kingdoms: IDOs of Basidiomycota.

    Science.gov (United States)

    Yuasa, Hajime J; Ball, Helen J

    2013-07-01

    Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and is found in animals, fungi and bacteria. In fungi, its primary role is to supply nicotinamide adenine dinucleotide (NAD(+)) via the kynurenine pathway. A number of organisms possess more than one IDO gene, for example, mammals have IDO1 and IDO2 genes. We previously reported that the Pezizomycotina fungi commonly possess three types of IDO genes, IDOα, IDOβ and IDOγ. In this study, we surveyed the nature of IDO genes from Basidiomycota fungi, which are categorized into three subphyla (Agaricomycotina, Pucciniomycotina and Ustilaginomycotina). The Agaricomycotina fungi generally have three types of IDO genes (IDOa, IDOb and IDOc), which are distinct from Pezizomycotina three isozymes. Pucciniomycotina and Ustilaginomycotina species possess two types of IDO; one forms a monophyletic clade with Agaricomycotina IDOs in the phylogenetic tree, these IDOs are referred to as "typical Basidiomycota IDOs". The other is IDOγ, which showed more than 40% identity with Pezizomycotina and ciliate IDOγ. We previously demonstrated that IDO2 in mammals and IDOγ in Perzizomycotina fungi have much lower catalytic efficiencies in an in vitro assay, compared with the other IDO isoforms found in the respective species. We have developed a functional assay to determine whether particular IDO enzymes have sufficient enzymatic activity to rescue a yeast strain where IDO-deletion has rendered it auxotrophic for nicotinic acid. IDOα and IDOβ showed comparable catalytic efficiency, both of them could function in the Pezizomycotina fungal L-Trp metabolism. The catalytic efficiency and functional capacity of the Basidiomycota IDOa and IDOb were similar to Pezizomycotina IDOα/IDOβ. We found that Basidiomycota IDOc could not rescue the nicotinic acid auxotroph, similar to other IDO enzymes with low catalytic efficiency (mammalian IDO2 and most fungal IDOγ). Our study suggests that some fungal IDO enzymes function in

  1. Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a ...

    African Journals Online (AJOL)

    Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a) Levels In Nigerian Subjects With and Without Coronary Heart Disease. ... Plasma lipoprotein (a) Concentrations and apo(a) isoforms were determined in 101 ... Article Metrics.

  2. Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein.

    Science.gov (United States)

    Su, C; Oliw, E H

    1996-06-14

    The fungus Gaeumannomyces graminis, which causes the major root disease of wheat known as "take-all," can metabolize linoleic acid to (8R)-hydroperoxylinoleic acid. The enzyme linoleate 8-dioxygenase abstracts hydrogen and introduces molecular oxygen in an antarafacial way at C-8. We have now purified the enzyme 1000-fold to a specific activity of 1.8 micronol/min/mg of protein. Acetone powder of mycelia of G. graminis was subjected to extraction and ammonium sulfate precipitation with solubilization. The 8-dioxygenase was purified by hydrophobic interaction chromatography, size-exclusion chromatography, anion-exchange chromatography, and immobilized metal ion affinity chromatography. The active enzyme appeared to consist of four subunits since the active enzyme had an apparent molecular mass of 520 kDa determined by gel filtration, while SDS-polyacrylamide gel electrophoresis showed a protein band of 130 kDa. Spectroscopy indicated the presence of heme. The characteristic pyridine ferrohemochrome alpha-band was found at 557 nm and the beta-band at 525 nm. The purified protein showed an absorption maximum at 408 nm (gamma, Soret). The absorption maximum shifted to 429 nm after reduction with dithionite and to 421 nm after treatment of the reduced enzyme with carbon monoxide. BW A4C, a hydroxamic acid derivative, inhibited the enzyme by >90% at 10 microM. The pH optimum was 7.2-7.4, the isoelectric point was 5.2 by chromatofocusing, and the Km values were 8 microM for linoleic acid and 30 microM for oxygen. We conclude that linoleate 8-dioxygenase appears to be a tetrameric hemoprotein distinct from other fatty-acid dioxygenases.

  3. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    KAUST Repository

    Frusciante, Sarah

    2014-08-05

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

  4. Ring-hydroxylating dioxygenases involved in PAH biodegradation : structure, function, biodiversity

    OpenAIRE

    Jouanneau, Yves; Martin, Florence; Krivobok, Serge; Willison, John Christopher

    2011-01-01

    International audience; The first step in the biodegradation of PAHs by aerobic bacteria is catalyzed by metalloenzymes known as ring-hydroxylating dioxygenases (RHDs). Because of the hydrophobic nature and chemical resistance of PAHs, their initial attack by RHDs is a difficult reaction, which is critical to the whole degradation process. This chapter gives an overview of the current knowledge on the genetics, structure, catalytic mechanism and diversity of RHDs involved in PAH degradation. ...

  5. 4-Hydroxyphenylpyruvate dioxygenase inhibitors in combination with safeners: solutions for modern and sustainable agriculture.

    Science.gov (United States)

    Ahrens, Hartmut; Lange, Gudrun; Müller, Thomas; Rosinger, Chris; Willms, Lothar; van Almsick, Andreas

    2013-09-01

    Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) prevent plant carotenoid pigment formation, which in turn leads to chlorophyll degradation. This "bleaching" herbicide mode of action provides weed-control products for various crops, such as rice, corn, and cereals. Combinations with suitable safeners allow the full exploitation of the potential of this compound class to selectively control major weed problems, including rapidly increasing cases of resistance against other important herbicide classes.

  6. Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice.

    Science.gov (United States)

    Ball, Helen J; Sanchez-Perez, Angeles; Weiser, Silvia; Austin, Christopher J D; Astelbauer, Florian; Miu, Jenny; McQuillan, James A; Stocker, Roland; Jermiin, Lars S; Hunt, Nicholas H

    2007-07-01

    Indoleamine 2,3-dioxygenase (INDO) and tryptophan 2,3-dioxygenase (TDO) each catalyze the first step in the kynurenine pathway of tryptophan metabolism. We describe the discovery of another enzyme with this activity, indoleamine 2,3-dioxygenase-like protein (INDOL1), which is closely related to INDO and is expressed in mice and humans. The corresponding genes have a similar genomic structure and are situated adjacent to each other on human and mouse chromosome 8. They are likely to have arisen by gene duplication before the origin of the tetrapods. The expression of INDOL1 is highest in the mouse kidney, followed by epididymis, and liver. Expression of mouse INDOL1 was further localized to the tubular cells in the kidney and the spermatozoa. INDOL1 was assigned its name because of its structural similarity to INDO. We demonstrate that INDOL1 catalyses the conversion of tryptophan to kynurenine therefore a more appropriate nomenclature for the enzymes might be INDO-1 and INDO-2, or the more commonly-used abbreviations, IDO-1 and IDO-2. Although the two proteins have similar enzymatic activities, their different expression patterns within tissues and during malaria infection, suggests a distinct role for each protein. This identification of INDOL1 may help to explain the regulation of the diversity of physiological and patho-physiological processes in which the kynurenine pathway is involved.

  7. Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01.

    Science.gov (United States)

    Dong, Xuesong; Fushinobu, Shinya; Fukuda, Eriko; Terada, Tohru; Nakamura, Shugo; Shimizu, Kentaro; Nojiri, Hideaki; Omori, Toshio; Shoun, Hirofumi; Wakagi, Takayoshi

    2005-04-01

    The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.

  8. [Isolation, charcaterization of an anthracene degrading bacterium Martelella sp. AD-3 and cloning of dioxygenase gene].

    Science.gov (United States)

    Cui, Chang-Zheng; Feng, Tian-Cai; Yu, Ya-Qi; Dong, Fei; Yang, Xin-Mei; Feng, Yao-Yu; Liu, Yong-Di; Lin, Han-Ping

    2012-11-01

    Anthracene, among the 16 US EPA polycyclic aromatic hydrocarbons (PAHs), is a typical low molecular weight environmental contaminant, which gains concern on its biodegradation under hypersaline condition. In this study, an anthracene-degrading bacterial strain was isolated from highly saline petroleum-contaminated soil. Based on its physiological, biochemical characteristics and 16S rDNA sequence analysis, the bacteria was preliminary identified and named as Martelella sp. AD-3. The strain was able to utilize anthracene as sole carbon source for growth and the degradation occurred under broad salinities (0.1% to 10%) and varying pHs (6.0 to 10.0). The optimized degradation conditions were initial concentration 25 mg x L(-1), culture temperature 30 degrees C, pH 9.0 and salinity 3%. And 94.6% of anthracene was degraded by strain AD-3 under the optimal conditions within 6 days. Degenerate primers design was performed with a reported dioxygenase alpha subunit homologous gene. A length of 307 bp fragment of the partial dioxygenase gene sequences (GenBank accession: JF823991.1) was amplified by nested PCR. The clones amino acid sequence from strain AD-3 showed 95% identity to that of the partial naphthalene dioxygenase large-subunit from Marinobacter sp. NCE312 (AF295033). The results lay a foundation for the further study of molecular mechanism involved in the PAHs biodegradation by strain AD-3.

  9. Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy.

    Science.gov (United States)

    Tiranti, Valeria; Viscomi, Carlo; Hildebrandt, Tatjana; Di Meo, Ivano; Mineri, Rossana; Tiveron, Cecilia; Levitt, Michael D; Prelle, Alessandro; Fagiolari, Gigliola; Rimoldi, Marco; Zeviani, Massimo

    2009-02-01

    Ethylmalonic encephalopathy is an autosomal recessive, invariably fatal disorder characterized by early-onset encephalopathy, microangiopathy, chronic diarrhea, defective cytochrome c oxidase (COX) in muscle and brain, high concentrations of C4 and C5 acylcarnitines in blood and high excretion of ethylmalonic acid in urine. ETHE1, a gene encoding a beta-lactamase-like, iron-coordinating metalloprotein, is mutated in ethylmalonic encephalopathy. In bacteria, ETHE1-like sequences are in the same operon of, or fused with, orthologs of TST, the gene encoding rhodanese, a sulfurtransferase. In eukaryotes, both ETHE1 and rhodanese are located within the mitochondrial matrix. We created a Ethe1(-/-) mouse that showed the cardinal features of ethylmalonic encephalopathy. We found that thiosulfate was excreted in massive amounts in urine of both Ethe1(-/-) mice and humans with ethylmalonic encephalopathy. High thiosulfate and sulfide concentrations were present in Ethe1(-/-) mouse tissues. Sulfide is a powerful inhibitor of COX and short-chain fatty acid oxidation, with vasoactive and vasotoxic effects that explain the microangiopathy in ethylmalonic encephalopathy patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase. Sulfur dioxygenase activity was absent in Ethe1(-/-) mice, whereas it was markedly increased by ETHE1 overexpression in HeLa cells and Escherichia coli. Therefore, ETHE1 is a mitochondrial sulfur dioxygenase involved in catabolism of sulfide that accumulates to toxic levels in ethylmalonic encephalopathy.

  10. Expression Pattern and Clinicopathological Relevance of the Indoleamine 2,3-Dioxygenase 1/Tryptophan 2,3-Dioxygenase Protein in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    I-Chien Chen

    2016-01-01

    Full Text Available Aims. Cancer cells use the indoleamine 2,3-dioxygenase 1 (IDO1 pathway to suppress the host’s immune response in order to facilitate survival, growth, invasion, and metastasis of malignant cells. Higher IDO1 expression was shown to be involved in colorectal cancer (CRC progression and to be correlated with impaired clinical outcome. However, the potential correlation between the expression of IDO1 in a CRC population with a low mutation rate of the APC gene remains unknown. Material and Methods. Tissues and blood samples were collected from 192 CRC patients. The expressions of IDO1, tryptophan 2,3-dioxygenase (TDO2, and beta-catenin proteins were analyzed by immunohistochemistry. Microsatellite instability (MSI was determined by PCR amplification of microsatellite loci. Results. The results showed that high IDO1 or TDO2 protein expression was associated with characteristics of more aggressive phenotypes of CRC. For the first time, they also revealed a positive correlation between the abnormal expression of beta-catenin and IDO1 or TDO2 proteins in a CRC population with a low mutation rate of APC. Conclusion. We concluded that an IDO1-regulated molecular pathway led to abnormal expression of beta-catenin in the nucleus/cytoplasm of CRC patients with low mutation rate of APC, making IDO1 an interesting target for immunotherapy in CRC.

  11. Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, M.; Khanna, S. [NIIT Univ, Neemrana (India). Dept. of Biotechnology & Bioinformation

    2010-04-15

    In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available. Coal-tar-contaminated soil was collected, which consisted of 122-122.5 mg g{sup -1} total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (I) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center {alpha}-subunit) common to all PAH dioxygenase enzymes and (iii) {beta}-subunit of dioxygenase. Phylotypes related to Proteobacteria ({Alpha}-, {Epsilon}- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of alpha-subunit and beta-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type (2Fe2S) cluster binding site suggested that these gene fragments encode for {alpha}-subunit of dioxygenase gene. Sequencing of the cloned libraries representing {alpha}-subunit gene fragments (Rf1) and beta-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil.

  12. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  13. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have u...... synthesized cohorts of pumps from the Golgi apparatus to the plasma membrane....

  14. FSH isoform pattern in classic galactosemia.

    Science.gov (United States)

    Gubbels, Cynthia S; Thomas, Chris M G; Wodzig, Will K W H; Olthaar, André J; Jaeken, Jaak; Sweep, Fred C G J; Rubio-Gozalbo, M Estela

    2011-04-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

  15. Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex.

    Science.gov (United States)

    Peng, Rihe; Fu, Xiaoyan; Tian, Yongsheng; Zhao, Wei; Zhu, Bo; Xu, Jing; Wang, Bo; Wang, Lijuan; Yao, Quanhong

    2014-11-01

    The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2-4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography-mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  16. Anaerobic enzyme·substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase

    NARCIS (Netherlands)

    Steiner, Roberto A.; Kalk, Kor H.; Dijkstra, Bauke W.

    2002-01-01

    Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic pr

  17. Anaerobic enzyme-substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase

    NARCIS (Netherlands)

    Steiner, RA; Kalk, KH; Dijkstra, BW

    2002-01-01

    Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic pr

  18. Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes.

    Science.gov (United States)

    Takami, Wako; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

    1999-04-01

    In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

  19. Mechanism of S-oxygenation by a cysteine dioxygenase model complex

    Science.gov (United States)

    Sastry, G. Narahari

    2012-01-01

    In this work we present the first computational study on a biomimetic cysteine dioxygenase model complex, [FeII(LN3S)]+ where LN3S is a tetradentate ligand with a bis(imino)pyridyl scaffold and a pendant arylthiolate group. The reaction mechanism of sulfur dioxygenation with O2 was examined by density functional theory (DFT) methods, and compared to results obtained for cysteine dioxygenase. The reaction proceeds via multistate reactivity patterns on competing singlet, triplet and quintet spin state surfaces. The reaction mechanism is analogous to that found for cysteine dioxygenase enzymes [Kumar, D.; Thiel, W.; de Visser, S. P. J. Am. Chem. Soc. 2011, 133, 3869–3882], hence the computations indicate that this complex can closely mimic the enzymatic process. The catalytic mechanism starts from an iron(III)-superoxo complex and the attack of the terminal oxygen atom of the superoxo group on the sulfur atom of the ligand. Subsequently, the dioxygen bond breaks to form an iron(IV)-oxo complex with a bound sulfenato group. After reorganization the second oxygen atom is transferred to the substrate to give a sulfinic acid product. An alternative mechanism involving the direct attack of dioxygen on the sulfur, without involving any iron-oxygen intermediates, was also examined. Importantly, a significant energetic preference for dioxygen coordinating to the iron center prior to attack at sulfur was discovered and serves to elucidate the function of the metal ion in the reaction process. The computational results are in good agreement with experimental observations, and the differences and similarities of the biomimetic complex and the enzymatic CDO center are highlighted. PMID:22091701

  20. The Targeting of Indoleamine 2,3 Dioxygenase -Mediated Immune Escape in Cancer

    DEFF Research Database (Denmark)

    Iversen, Trine Zeeberg; Andersen, Mads Hald; Svane, Inge Marie

    2015-01-01

    The era of immunotherapies was unleashed in 2010 with the Food and Drug Administration (FDA) approval of the first therapeutic vaccine sipuleucel-T as a standard treatment for metastatic prostate cancer. Next, the first immune-activating anticytotoxic lymphocyte antigen-4 (CTLA-4) antibody...... a peptide vaccination with a HLA-A2-restricted epitope derived from indoleamine 2,3 dioxygenase (IDO). The overall aim in this trial was to evaluate safety and tolerability of IDO as an anticancer vaccine target in patients with NSCLC and to assess whether immunity correlated to clinical response....

  1. Iron(III) complexes of certain tetradentate phenolate ligands as functional models for catechol dioxygenases

    Indian Academy of Sciences (India)

    Mallayan Palaniandavar; Marappan Velusamy; Ramasamy Mayilmurugan

    2006-11-01

    Catechol 1,2-dioxygenase (CTD) and protocatechuate 3,4-dioxygenase (PCD) are bacterial non-heme iron enzymes, which catalyse the oxidative cleavage of catechols to cis, cis-muconic acids with the incorporation of molecular oxygen via a mechanism involving a high-spin ferric centre. The iron(III) complexes of tripodal phenolate ligands containing N3O and N2O2 donor sets represent the metal binding region of the iron proteins. In our laboratory iron(III) complexes of mono- and bisphenolate ligands have been studied successfully as structural and functional models for the intradiol-cleaving catechol dioxygenase enzymes. The single crystal X-ray crystal structures of four of the complexes have been determined. One of the bis-phenolato complexes contains a FeN2O2Cl chromophore with a novel trigonal bipyramidal coordination geometry. The Fe-O-C bond angle of 136.1° observed for one of the iron(III) complex of a monophenolate ligand is very similar to that in the enzymes. The importance of the nearby sterically demanding coordinated -NMe2 group has been established and implies similar stereochemical constraints from the other ligated amino acid moieties in the 3,4-PCD enzymes, the enzyme activity of which is traced to the difference in the equatorial and axial Fe-O(tyrosinate) bonds (Fe-O-C, 133, 148°). The nature of heterocyclic rings of the ligands and the methyl substituents on them regulate the electronic spectral features, FeIII/FeII redox potentials and catechol cleavage activity of the complexes. Upon interacting with catecholate anions, two catecholate to iron(III) charge transfer bands appear and the low energy band is similar to that of catechol dioxygenase-substrate complex. Four of the complexes catalyze the oxidative cleavage of H2DBC by molecular oxygen to yield intradiol cleavage products. Remarkably, the more basic N-methylimidazole ring in one of the complexes facilitates the rate-determining productreleasing phase of the catalytic reaction. The present

  2. Compound-Specific Isotope Analysis of Nitroaromatic Contaminant Transformations by Nitroarene Dioxygenases

    Science.gov (United States)

    Pati, Sarah G.; Kohler, Hans-Peter E.; Hofstetter, Thomas B.

    2014-05-01

    Dioxygenation is an important biochemical reaction that often initiates the mineralization of recalcitrant organic contaminants such as nitroaromatic explosives, chlorinated benzenes, and polycyclic aromatic hydrocarbons. However, to assess the extent of dioxygenation in contaminated environments is difficult because of competing transformation processes and further reactions of the dioxygenation products. Compound-specific isotope analysis (CSIA) offers a new approach to reliably quantify biodegradation initiated by dioxygenation based on changes in stable isotope ratios of the pollutant. For CSIA it is essential to know the kinetic isotope effects (KIEs) pertinent to the dioxygenation mechanism of organic contaminants. Unfortunately, the range of KIEs of such reactions is poorly constrained although many dioxygenase enzymes with a broad substrate specificity have been reported. Dioxygenase enzymes usually exhibit complex reaction kinetics involving multiple substrates and substrate-specific binding modes which makes the determination of KIEs challenging. The goal of this study was to explore the magnitude and variability of 13C-, 2H-, and 15N-KIEs for the dioxygenation of one contaminant class, that is nitroaromatic contaminants (NACs). To this end, we investigated the C, H, and N isotope fractionation during the dioxygenation of nitrobenzene (NB), 2-nitrotoluene (2-NT), and 3-nitrotoluene (3-NT) by pure cultures, E. coli clones, cell extracts, and purified enzymes. From isotope fractionations measured in the substrates and reaction products, we determined dioxygenation KIEs for different combinations of the three substrates with nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO). The 13C-, 2H-, and 15N-KIEs for the dioxygenation of NB by NBDO were consistent for all experimental systems considered (i.e., Comamonas sp. Strain JS765, E. coli clones, cell extracts of E. coli clones, and purified NBDO). This observation suggests that the isotope

  3. Structure of the 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    Energy Technology Data Exchange (ETDEWEB)

    Keegan, R.; Lebedev, A. [RAL, Harwell Oxford, Didcot OX11 0FA (United Kingdom); Erskine, P.; Guo, J.; Wood, S. P. [UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF (United Kingdom); Hopper, D. J. [Aberystwyth University, Penglais, Aberystwyth SY23 3DA Wales (United Kingdom); Rigby, S. E. J. [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Cooper, J. B., E-mail: jon.cooper@ucl.ac.uk [UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF (United Kingdom); RAL, Harwell Oxford, Didcot OX11 0FA (United Kingdom)

    2014-09-01

    The first X-ray structure of a 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP at a resolution of 2.2 Å is reported. This structure establishes that the enzyme adopts the cupin-fold, forming compact dimers with a pronounced hydrophobic interface between the monomers. Each monomer possesses a catalytic ferrous iron that is coordinated by three histidines (76, 78 and 114) and an additional ligand which has been putatively assigned as a carbonate, although formate and acetate are possibilities. The enzyme 2, 4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2, 4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in

  4. A synthetic model of the putative Fe(II)-iminobenzosemiquinonate intermediate in the catalytic cycle of o-aminophenol dioxygenases.

    Science.gov (United States)

    Bittner, Michael M; Lindeman, Sergey V; Fiedler, Adam T

    2012-03-28

    The oxidative ring cleavage of aromatic substrates by nonheme Fe dioxygenases is thought to involve formation of a ferrous-(substrate radical) intermediate. Here we describe the synthesis of the trigonal-bipyramdial complex Fe((Ph2)Tp)(ISQ(tBu)) (2), the first synthetic example of an iron(II) center bound to an iminobenzosemiquinonate (ISQ) radical. The unique electronic structure of this S = 3/2 complex and its one-electron oxidized derivative ([3](+)) have been established on the basis of crystallographic, spectroscopic, and computational analyses. These findings further demonstrate the viability of Fe(2+)-ISQ intermediates in the catalytic cycles of o-aminophenol dioxygenases.

  5. Expression of Contractile Protein Isoforms in Microgravity

    Science.gov (United States)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  6. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  7. The landscape of isoform switches in human cancers

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

    2017-01-01

    developed methods for identification and visualization of isoform switches with predicted functional consequences. Using these methods, we characterized isoform switching in RNA-seq data from >5,500 cancer patients covering 12 solid cancer types. Isoform switches with potential functional consequences were...... highly predictive of patient survival independent of cancer types. Our data constitute an important resource for cancer researchers, available through interactive web tools. Moreover, our methods, available as an R package, enable systematic analysis of isoform switches from other RNA-seq datasets...

  8. The role of indoleamine 2,3-dioxygenase in a mouse model of neuroinflammation-induced depression

    NARCIS (Netherlands)

    Dobos, Nikoletta; de Vries, Erik F.J.; Kema, Ido P.; Patas, Konstantinos; Prins, Marloes; Nijholt, Ingrid M.; Dierckx, Rudi A.; Korf, Jakob; den Boer, Johan A.; Luiten, Paul G M; Eisel, Ulrich L M; Smith, Gwenn S.

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO), an enzyme which is activated by pro-inflammatory cytokines, has been suggested as a potential link between neuroinflammatory processes in neurodegenerative diseases (like Alzheimer's disease) and depression. The present study aimed to determine whether

  9. The Role of Indoleamine 2,3-Dioxygenase in a Mouse Model of Neuroinflammation-Induced Depression

    NARCIS (Netherlands)

    Dobos, Nikoletta; de Vries, Erik F. J.; Kema, Ido P.; Patas, Konstantinos; Prins, Marloes; Nijholt, Ingrid M.; Dierckx, Rudi A.; Korf, Jakob; den Boer, Johan A.; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Borsello, Tiziana

    2012-01-01

    Indoleamine 2,3-dioxygenase (IDO), an enzyme which is activated by pro-inflammatory cytokines, has been suggested as a potential link between neuroinflammatory processes in neurodegenerative diseases (like Alzheimer's disease) and depression. The present study aimed to determine whether neuroinflamm

  10. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    Science.gov (United States)

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  11. Tocopherol isoforms in parenteral lipid emulsions and neutrophil activation.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Beunk, J.; Naber, A.H.J.; Swinkels, D.W.

    2002-01-01

    BACKGROUND AND AIMS: Tocopherol is a lipid-soluble anti-oxidant that exists in several isoforms. Patients on total parenteral nutrition depend on lipid emulsions for their tocopherol intake. In the present study, we analysed the content of tocopherol isoforms in various lipid emulsions. We also

  12. Evidence for leptin receptor isoforms heteromerization at the cell surface.

    Science.gov (United States)

    Bacart, Johan; Leloire, Audrey; Levoye, Angélique; Froguel, Philippe; Jockers, Ralf; Couturier, Cyril

    2010-06-01

    Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

  13. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  14. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    National Research Council Canada - National Science Library

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    .... There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms...

  15. Engineering of insulin receptor isoform-selective insulin analogues.

    Directory of Open Access Journals (Sweden)

    Tine Glendorf

    Full Text Available BACKGROUND: The insulin receptor (IR exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.

  16. Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type III.

    Science.gov (United States)

    Rüetschi, U; Cerone, R; Pérez-Cerda, C; Schiaffino, M C; Standing, S; Ugarte, M; Holme, E

    2000-06-01

    Tyrosinemia type III (OMIM 276710) is an autosomal recessive disorder caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPD), the second enzyme in the tyrosine catabolic pathway. The enzyme deficiency results in an accumulation and increased excretion of tyrosine and phenolic metabolites. Only a few cases with the disorder have been described, and the clinical spectrum of the disorder is unknown. Reported patients have presented with mental retardation or neurological symptoms or have been picked up by neonatal screening. We have identified four presumed pathogenic mutations (two missense and two nonsense mutations) in the HPD gene in three unrelated families encompassing four homozygous individuals and one compound heterozygous individual with tyrosinemia type III. Furthermore, a number of polymorphic mutations have been identified in the HPD gene. No correlation of the severity of the mutation and enzyme deficiency and mental function has been found; neither do the recorded tyrosine levels correlate with the clinical phenotype.

  17. Characterization of catechol 1,2-dioxygenase from cell extracts of Sphingomonas xenophaga QYY

    Institute of Scientific and Technical Information of China (English)

    GOU Min; QU YuanYuan; ZHOU JiTi; LI Ang; M.Salah Uddin

    2009-01-01

    Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C120) in cell extracts. Characterization of the crude C120 showed that the maximum activity was obtained at 40-70℃ and pH 7.8-8.8. Metal ions had different influences on the activity of crude C120. It was suggested that strain QYY possessed an inducible and ferric-dependent C120. Kinetic studies showed that the value of Vmax and Km was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C120 was achieved by a HiTrap Q Sepharose column chromatography.

  18. Characterization of catechol 1,2-dioxygenase from cell extracts of Sphingomonas xenophaga QYY

    Institute of Scientific and Technical Information of China (English)

    M.Salah; Uddin

    2009-01-01

    Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. Characterization of the crude C12O showed that the maximum activity was obtained at 40-70℃ and pH 7.8-8.8. Metal ions had different influences on the activity of crude C12O. It was suggested that strain QYY possessed an inducible and ferric-dependent C12O. Kinetic studies showed that the value of Vmax and Km was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C12O was achieved by a HiTrap Q Sepharose column chromatography.

  19. Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators

    DEFF Research Database (Denmark)

    Sørensen, Rikke Bæk; Hadrup, Sine Reker; Svane, Inge Marie

    2011-01-01

    Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We...... show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO+ suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore......, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-alpha while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate- guanosine, soluble cytotoxic T lymphocyte-associated antigen 4...

  20. Synthesis and bioevaluation of pyrazole-benzimidazolone hybrids as novel human 4-Hydroxyphenylpyruvate dioxygenase inhibitors.

    Science.gov (United States)

    Xu, Yu-Ling; Lin, Hong-Yan; Ruan, Xu; Yang, Sheng-Gang; Hao, Ge-Fei; Yang, Wen-Chao; Yang, Guang-Fu

    2015-03-06

    4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme in tyrosine catabolism, is an important target for treating type I tyrosinemia. Inhibition of HPPD can effectively alleviate the symptoms of type I tyrosinemia. However, only one commercial HPPD inhibitor, 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC), has been available for clinical use so far. In the present study, a series of novel pyrazole-benzimidazolone hybrids were designed, synthesized and evaluated as potent human HPPD inhibitors. Most of the new compounds displayed significant inhibitory activity against the recombinant human HPPD. Moreover, compound 9l was identified as the most potent candidate with IC50 value of 0.021 μM against recombinant human HPPD, about 3-fold more potent than NTBC. Thus the pyrazole-benzimidazolone hybrid has great potential to be further developed for the treatment of type I tyrosinemia.

  1. Degradation of phenanthrene and pyrene using genetically engineered dioxygenase producing Pseudomonas putida in soil

    Directory of Open Access Journals (Sweden)

    Mardani Gashtasb

    2016-01-01

    Full Text Available Bioremediation use to promote degradation and/or removal of contaminants into nonhazardous or less-hazardous substances from the environment using microbial metabolic ability. Pseudomonas spp. is one of saprotrophic soil bacterium and can be used for biodegradation of polycyclic aromatic hydrocarbons (PAHs but this activity in most species is weak. Phenanthrene and pyrene could associate with a risk of human cancer development in exposed individuals. The aim of the present study was application of genetically engineered P. putida that produce dioxygenase for degradation of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC method. The nahH gene that encoded catechol 2,3-dioxygenase (C23O was cloned into pUC18 and pUC18-nahH recombinant vector was generated and transformed into wild P. putida, successfully. The genetically modified and wild types of P. putida were inoculated in soil and pilot plan was prepared. Finally, degradation of phenanthrene and pyrene by this bacterium in spiked soil were evaluated using HPLC measurement technique. The results were showed elimination of these PAH compounds in spiked soil by engineered P. putida comparing to dishes containing natural soil with normal microbial flora and inoculated autoclaved soil by wild type of P. putida were statistically significant (p0.05 but it was few impact on this process (more than 2%. Additional and verification tests including catalase, oxidase and PCR on isolated bacteria from spiked soil were indicated that engineered P. putida was alive and functional as well as it can affect on phenanthrene and pyrene degradation via nahH gene producing. These findings indicated that genetically engineered P. putida generated in this work via producing C23O enzyme can useful and practical for biodegradation of phenanthrene and pyrene as well as petroleum compounds in polluted environments.

  2. 2,3-Dihydroxybiphenyl dioxygenase gene was first discovered in Arthrobacter sp. strain P J3

    Institute of Scientific and Technical Information of China (English)

    YANG MeiYing; MA PengDa; LI WenMing; LIU JinYing; LI Liang; ZHU XiaoJuan; WANG XingZhi

    2007-01-01

    Bacterium strain PJ3, isolated from wastewater and identified as Arthrobacter sp. bacterium based on its 16S rDNA gene, could use carbazole as the sole carbon, nitrogen and energy source. The genomic libraryof strain PJ3 was constructed and a positive clone JM109 (pUCW402) was screened out for the expression of dioxygenase by the ability to form yellow ring-fission product. A 2,3-dihydroxybiphenyl dioxygenase (23DHBD) gene of 933 bp was found in the 3360 bp exogenous fragment of pUCW402 by GenSCAN software and BLAST analysis. The phylogenetic analysis showed that 23DHBD from strain PJ3 formed a deep branch separate from a cluster containing most known 23DHBD in GenBank.Southern hybridization confirmed for the first time that the 23DHBD gene was from the genomic DNA of Arthrobacter sp. PJ3. In order to test the gene function, recombinant bacterium BL21 (pETW-8) was constructed to express 23DHBD. The expression level in BL21 (pETW-8) was highest compared with the recombinant bacteria JM109 (pUCW402) and strain PJ3. We observed that 23DHBD was not absolute specific. The enzyme activity was higher with 2,3-dihydroxybiphenyl as a substrate than with catechol.The substrate specificity assay suggested that 23DHBD was essential for cleavage of bi-cyclic aromatic compounds during the course of aromatic compound biodegradation in Arthrobacter sp. strain PJ3.

  3. Promotion of Germination Using Hydroxamic Acid Inhibitors of 9-cis-Epoxycarotenoid Dioxygenase

    Science.gov (United States)

    Awan, Sajjad Z.; Chandler, Jake O.; Harrison, Peter J.; Sergeant, Martin J.; Bugg, Timothy D. H.; Thompson, Andrew J.

    2017-01-01

    Abscisic acid (ABA) inhibits seed germination and the regulation of ABA biosynthesis has a role in maintenance of seed dormancy. The key rate-limiting step in ABA biosynthesis is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Two hydroxamic acid inhibitors of carotenoid cleavage dioxygenase (CCD), D4 and D7, previously found to inhibit CCD and NCED in vitro, are shown to have the novel property of decreasing mean germination time of tomato (Solanum lycopersicum L.) seeds constitutively overexpressing LeNCED1. Post-germination, D4 exhibited no negative effects on tomato seedling growth in terms of height, dry weight, and fresh weight. Tobacco (Nicotiana tabacum L.) seeds containing a tetracycline-inducible LeNCED1 transgene were used to show that germination could be negatively and positively controlled through the chemical induction of gene expression and the chemical inhibition of the NCED protein: application of tetracycline increased mean germination time and delayed hypocotyl emergence in a similar manner to that observed when exogenous ABA was applied and this was reversed by D4 when NCED expression was induced at intermediate levels. D4 also improved germination in lettuce (Lactuca sativa L.) seeds under thermoinhibitory temperatures and in tomato seeds imbibed in high osmolarity solutions of polyethylene glycol. D4 reduced ABA and dihydrophaseic acid accumulation in tomato seeds overexpressing LeNCED1 and reduced ABA accumulation in wild type tomato seeds imbibed on polyethylene glycol. The evidence supports a mode of action of D4 through NCED inhibition, and this molecule provides a lead compound for the design of NCED inhibitors with greater specificity and potency.

  4. Adiponectin isoforms: a potential therapeutic target in rheumatoid arthritis?

    Science.gov (United States)

    Frommer, Klaus W; Schäffler, Andreas; Büchler, Christa; Steinmeyer, Jürgen; Rickert, Markus; Rehart, Stefan; Brentano, Fabia; Gay, Steffen; Müller-Ladner, Ulf; Neumann, Elena

    2012-10-01

    Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage. Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay. In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes. The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.

  5. Nonmuscle myosin II isoforms coassemble in living cells.

    Science.gov (United States)

    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

  6. p53 Family: Role of Protein Isoforms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  7. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-11-17

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

  8. Structure of the Dioxygenase AsqJ: Mechanistic Insights into a One-Pot Multistep Quinolone Antibiotic Biosynthesis

    KAUST Repository

    Bräuer, Alois

    2015-11-10

    © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Multienzymatic cascades are responsible for the biosynthesis of natural products and represent a source of inspiration for synthetic chemists. The FeII/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans is outstanding because it stereoselectively catalyzes both a ferryl-induced desaturation reaction and epoxidation on a benzodiazepinedione. Interestingly, the enzymatically formed spiro epoxide spring-loads the 6,7-bicyclic skeleton for non-enzymatic rearrangement into the 6,6-bicyclic scaffold of the quinolone alkaloid 4′-methoxyviridicatin. Herein, we report different crystal structures of the protein in the absence and presence of synthesized substrates, surrogates, and intermediates that mimic the various stages of the reaction cycle of this exceptional dioxygenase.

  9. Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment.

    Science.gov (United States)

    Han, Hesong; Iakovenko, Liudmyla; Wilson, Adam C

    2015-01-01

    Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.

  10. Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment.

    Directory of Open Access Journals (Sweden)

    Hesong Han

    Full Text Available Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.

  11. Atom Tunneling in the Hydroxylation Process of Taurine/α-Ketoglutarate Dioxygenase Identified by Quantum Mechanics/Molecular Mechanics Simulations.

    Science.gov (United States)

    Álvarez-Barcia, Sonia; Kästner, Johannes

    2017-06-01

    Taurine/α-ketoglutarate dioxygenase is one of the most studied α-ketoglutarate-dependent dioxygenases (αKGDs), involved in several biotechnological applications. We investigated the key step in the catalytic cycle of the αKGDs, the hydrogen transfer process, by a quantum mechanics/molecular mechanics approach (B3LYP/CHARMM22). Analysis of the charge and spin densities during the reaction demonstrates that a concerted mechanism takes place, where the H atom transfer happens simultaneously with the electron transfer from taurine to the Fe═O cofactor. We found the quantum tunneling of the hydrogen atom to increase the rate constant by a factor of 40 at 5 °C. As a consequence, a quite high kinetic isotope effect close to 60 is obtained, which is consistent with the experimental value.

  12. HIV-1 Tat activates indoleamine 2,3 dioxygenase in murine organotypic hippocampal slice cultures in a p38 mitogen-activated protein kinase-dependent manner

    Directory of Open Access Journals (Sweden)

    Kelley Keith W

    2011-08-01

    Full Text Available Abstract Background We have established that activation of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO mediates the switch from cytokine-induced sickness behavior to depressive-like behavior. Because human immunodeficiency virus type 1 (HIV-1 Tat protein causes depressive-like behavior in mice, we investigated its ability to activate IDO in organotypic hippocampal slice cultures (OHSCs derived from neonatal C57BL/6 mice. Methods Depressive-like behavior in C57BL/6J mice was assessed by the forced swim test. Expression of cytokines and IDO mRNA in OHSCs was measured by real-time RT-PCR and cytokine protein was measured by enzyme-linked immunosorbent assays (ELISAs. p38 MAPK phosphorylation was analyzed by western blot. Results Intracerebroventricular (i.c.v. administration of Tat (40 ng induced depressive-like behavior in the absence of sickness. Addition of Tat (40 ng/slice to the medium of OHSCs induced IDO steady-state mRNA that peaked at 6 h. This effect was potentiated by pretreatment with IFNγ. Tat also induced the synthesis and release of TNFα and IL-6 protein in the supernatant of the slices and increased expression of the inducible isoform of nitric oxide synthase (iNOS and the serotonin transporter (SERT. Tat had no effect on endogenous synthesis of IFNγ. To explore the mechanisms of Tat-induced IDO expression, slices were pretreated with the p38 mitogen-activated protein kinase (MAPK inhibitor SB 202190 for 30 min before Tat treatment. SB 202190 significantly decreased IDO expression induced by Tat, and this effect was accompanied by a reduction of Tat-induced expression of TNFα, IL-6, iNOS and SERT. Conclusion These data establish that Tat induces IDO expression via an IFNγ-independent mechanism that depends upon activation of p38 MAPK. Targeting IDO itself or the p38 MAPK signaling pathway could provide a novel therapy for comorbid depressive disorders in HIV-1-infected patients.

  13. Central Administration of Lipopolysaccharide Induces Depressive-like Behavior in Vivo and Activates Brain Indoleamine 2,3 Dioxygenase In Murine Organotypic Hippocampal Slice Cultures

    Directory of Open Access Journals (Sweden)

    Kavelaars Annemieke

    2010-08-01

    Full Text Available Abstract Background Transient stimulation of the innate immune system by an intraperitoneal injection of lipopolysaccharide (LPS activates peripheral and central expression of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO which mediates depressive-like behavior. It is unknown whether direct activation of the brain with LPS is sufficient to activate IDO and induce depressive-like behavior. Methods Sickness and depressive-like behavior in C57BL/6J mice were assessed by social exploration and the forced swim test, respectively. Expression of cytokines and IDO mRNA was measured by real-time RT-PCR and cytokine protein was measured by enzyme-linked immunosorbent assays (ELISAs. Enzymatic activity of IDO was estimated as the amount of kynurenine produced from tryptophan as determined by high pressure liquid chromatography (HPLC with electrochemical detection. Results Intracerebroventricular (i.c.v. administration of LPS (100 ng increased steady-state transcripts of TNFα, IL-6 and the inducible isoform of nitric oxide synthase (iNOS in the hippocampus in the absence of any change in IFNγ mRNA. LPS also increased IDO expression and induced depressive-like behavior, as measured by increased duration of immobility in the forced swim test. The regulation of IDO expression was investigated using in situ organotypic hippocampal slice cultures (OHSCs derived from brains of newborn C57BL/6J mice. In accordance with the in vivo data, addition of LPS (10 ng/ml to the medium of OHSCs induced steady-state expression of mRNA transcripts for IDO that peaked at 6 h and translated into increased IDO enzymatic activity within 8 h post-LPS. This activation of IDO by direct application of LPS was preceded by synthesis and secretion of TNFα and IL-6 protein and activation of iNOS while IFNγ expression was undetectable. Conclusion These data establish that activation of the innate immune system in the brain is sufficient to activate IDO and induce

  14. Isoform-specific targeting of ROCK proteins in immune cells

    OpenAIRE

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform ...

  15. Neuronal profilin isoforms are addressed by different signalling pathways.

    Directory of Open Access Journals (Sweden)

    Kai Murk

    Full Text Available Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF, on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

  16. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  17. Yeast ADP/ATP Carrier Isoform 2

    Science.gov (United States)

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  18. Sequential oxygenation of linoleic acid in the fungus Gaeumannomyces graminis: stereochemistry of dioxygenase and hydroperoxide isomerase reactions.

    Science.gov (United States)

    Hamberg, M; Zhang, L Y; Brodowsky, I D; Oliw, E H

    1994-02-15

    Linoleic acid is sequentially oxygenated to (7S,8S)-dihydroxylinoleic acid by dioxygenase and hydroperoxide isomerase activities present in the fungus Gaeumannomyces graminis (Brodowsky, I. D., Hamberg, M., and Oliw, E. H., J. Biol. Chem. 267, 14738-14745 (1992)). Linoleic acids stereospecifically deuterated at C-7 and C-8 were prepared by biological desaturation of the corresponding stearates and used to determine the stereochemistry of the hydrogen abstractions occurring in the dioxygenase- and hydroperoxide isomerase-catalyzed reactions. The dioxygenase reaction was found to involve stereospecific abstraction of the pro-S hydrogen from C-8 followed by antarafacial insertion of dioxygen to produce (8R)-hydroperoxylinoleic acid. The hydroperoxide isomerase reaction consisted of conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid by stereospecific elimination of the pro-S hydrogen from C-7 and intramolecular suprafacial insertion of oxygen at C-7. Accordingly, during the conversion of linoleic acid into (8R)-hydroperoxylinoleic acid, the absolute configuration of C-8 was inverted, while the conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid occurred with retention of absolute configuration at C-7.

  19. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    Energy Technology Data Exchange (ETDEWEB)

    Turick, Charles E. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Beliaev, Alex S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Zakrajsek, Brian A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Reardon, Catherine L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lowy, Daniel A. [Nova Research Inc., Alexandria, VA (United States); Poppy, Tara E. [Univ. of South Carolina, Aiken, SC (United States); Maloney, Andrea [Winthrop Univ., Rock Hill, SC (United States); Ekechukwu, Amy A. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2009-05-01

    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.

  20. Crystallization and preliminary crystallographic analysis of 2-aminophenol 1,6-dioxygenase complexed with substrate and with an inhibitor.

    Science.gov (United States)

    Li, De-Feng; Zhang, Jia-Yue; Hou, Yanjie; Liu, Lei; Liu, Shuang-Jiang; Liu, Wei

    2012-11-01

    Dioxygen activation implemented by nonhaem Fe(II) enzymes containing the 2-His-1-carboxylate facial triad has been extensively studied in recent years. Extradiol dioxygenase is the archetypal member of this superfamily and catalyzes the oxygenolytic ring opening of catechol analogues. Here, the crystallization and preliminary X-ray analysis of 2-aminophenol 1,6-dioxygenase, an enzyme representing a minor subset of extradiol dioxygenases that catalyze the fission of 2-aminophenol rather than catecholic compounds, is reported. Crystals of the holoenzyme with FeII and of complexes with the substrate 2-aminophenol and the suicide inhibitor 4-nitrocatechol were grown using the cocrystallization method under the same conditions as used for the crystallization of the apoenzyme. The crystals belonged to space group C2 and diffracted to 2.3-2.7 Å resolution; the crystal that diffracted to the highest resolution had unit-cell parameters a=270.24, b=48.39, c=108.55 Å, β=109.57°. All X-ray data sets collected from diffraction-quality crystals were suitable for structure determination.

  1. The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation.

    Science.gov (United States)

    Hinz, U G; Fivaz, J; Girod, P A; Zyrd, J P

    1997-09-01

    Genomic and cDNA clones derived from the gene (dodA) coding for DOPA dioxygenase, a key enzyme in the betalain pathway, were obtained from the basidiomycete Amanita muscaria. A cDNA library was established in the phage lambda ZapII and dodA clones were isolated using polyclonal antibodies raised against the purified enzyme. Their identity was confirmed by comparison of the deduced amino acid sequence with the sequence of several tryptic peptide fragments of DOPA dioxygenase. The gene coded for a 228-amino acid protein that showed no homology to published sequences. The coding region was interrupted by five short introns. Regulation was shown to occur at the transcriptional level; the mRNA accumulated to high levels only in the coloured cap tissue. dodA was found to be a single-copy gene in A. muscaria. To our knowledge, this is the first gene from the betalain pathway to be cloned. It encodes a type of aromatic ring-cleaving dioxygenase that has not been previously described.

  2. Molecular mechanism of strict substrate specificity of an extradiol dioxygenase, DesB, derived from Sphingobium sp. SYK-6.

    Directory of Open Access Journals (Sweden)

    Keisuke Sugimoto

    Full Text Available DesB, which is derived from Sphingobium sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of S. sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4 of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3 of the gallate, is likely to serve as the catalytic base. Glu377' interacts with OH (5 of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.

  3. Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219

    Energy Technology Data Exchange (ETDEWEB)

    Sugimoto, Keisuke; Matsufuzi, Kazuki; Ohnuma, Hiroaki [Department of Material Chemistry, Asahikawa National College of Technology, 2-2-1-6 Shunko-dai, Asahikawa, Hokkaido 071-8142 (Japan); Senda, Miki [Japan Biological Information Research Center (JBIRC), Japan Biological Informatics Consortium (JBIC), 2-42 Aomi, Koto-ku, Tokyo 135-0064 (Japan); Fukuda, Masao [Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188 (Japan); Senda, Toshiya, E-mail: tsenda@jbirc.aist.go.jp [Biological Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-42 Aomi, Koto-ku, Tokyo (Japan); Department of Material Chemistry, Asahikawa National College of Technology, 2-2-1-6 Shunko-dai, Asahikawa, Hokkaido 071-8142 (Japan)

    2006-02-01

    PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, and diffracts to 2.3 Å resolution. Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.

  4. THE ROLE OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE IN ENHANCEMENT OF SOLID-PHASE ELECTRON TRANSFER BY SHEWANELLA ONEIDENSIS MR-1

    Energy Technology Data Exchange (ETDEWEB)

    Turick, C; Amy Ekechukwu, A

    2007-06-01

    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  5. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross...

  6. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

    Directory of Open Access Journals (Sweden)

    Kubo Takeo

    2010-02-01

    Full Text Available Abstract Background The ecdysone receptor (EcR regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. Results The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Conclusions Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional

  7. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    OpenAIRE

    Fagotti, A; Gabbiani, G.; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a stre...

  8. Characterization of arene di-oxygenases involved in polycyclic aromatic hydrocarbons biodegradation in Mycobacterium sp. 6PY1; Caracterisation d'arene dioxygenases impliquees dans la biodegradation des hydrocarbures aromatiques polycycliques chez Mycobacterium sp. 6PY1

    Energy Technology Data Exchange (ETDEWEB)

    Kuony, S.

    2005-06-15

    This thesis deals with the bacterial biodegradation of pollutants called polycyclic aromatic hydrocarbons (PAHs). The bacterium Mycobacterium sp. 6PY1 was isolated from a polluted soil for its ability to use pyrene, a 4-ring PAH, as sole source of carbon and energy. To learn about the pyrene metabolic pathway, the identification of the enzymes involved in this process has been undertaken using a proteomic approach. This approach revealed the occurrence of two ring-hydroxylating di-oxygenases in strain 6PY1, which could catalyze the initial attack of pyrene. The goal of this study was to clone the genes encoding the di-oxygenases identified in Mycobacterium sp. 6PY1, over-express these genes in an heterologous system in order to facilitate the purification of the corresponding enzymes, and determine the biochemical and catalytic properties of these enzymes. The pdoA1B1 genes encoding the terminal component of a di-oxygenase were cloned and over-expressed in Escherichia coli. The catalytic properties of this enzyme, called Pdo1, were determined in vivo by measuring the oxidation products of 2- to 4-ring PAHs by gas chromatography coupled to mass spectrometry (GC-MS). Analysis of the selectivity of the enzyme, as determined using GC-MS, showed that Pdo1 preferentially oxidized 3- or 4-ring PAHs, including phenanthrene and pyrene, but was inactive on di-aromatic compounds such as naphthalene and biphenyl. Pdo1 was unstable and was therefore purified in inactive form. The genes encoding a second di-oxygenase component were found in a locus containing two other catabolic genes. The pdoA2B2 genes encoded an enzyme called Pdo2 showing a narrow specificity towards 2- to 3-ring PAHs, and a high preference for phenanthrene. Pdo2 is an a3{beta}3 hexamer, containing [2Fe-2S] Rieske clusters which confer it a characteristic absorbance spectrum. A third set of genes possibly encoding another di-oxygenase was discovered in the genome of Mycobacterium sp. 6PY1. This set is closely

  9. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  10. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    Science.gov (United States)

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  11. Indoleamine 2,3-dioxygenase expression in patients with allergic rhinitis: a case-control study

    Directory of Open Access Journals (Sweden)

    Luukkainen Annika

    2011-12-01

    Full Text Available Abstract Background Indoleamine 2,3-dioxygenase (IDO is a tryptophan catalyzing enzyme. It has been suggested that it has a role in lower airway allergic inflammations, but its role in allergic rhinitis has not been investigated. Objective Our aim was to evaluate the expression of IDO in the nasal mucosa of allergic rhinitis patients allergic to birch pollen during peak exposure to birch pollen allergen and compare it to non-atopic patients. Methods IDO expression was immunohistochemically evaluated from nasal specimens obtained in- and off-season from otherwise healthy non-smoking volunteers both allergic to birch pollen (having mild or moderate allergic rhinoconjunctivitis and non-allergic controls. Results: The IDO expression levels were low in healthy controls and remained low also in patients allergic to birch pollen. There were no differences in the expression of IDO in- and off-season in either healthy or allergic subjects. Conclusions There is a controversy in the role of IDO in upper and lower airways during allergic airway disease. It seems that IDO is associated to allergic inflammations of the lower airways, but does not have a local role in the nasal cavity at least in mild or moderate forms of allergic rhinitis.

  12. Nanomedicine and cancer immunotherapy: focus on indoleamine 2,3-dioxygenase inhibitors

    Science.gov (United States)

    Zulfiqar, Bilal; Mahroo, Amnah; Nasir, Kaenat; Farooq, Rai Khalid; Jalal, Nasir; Rashid, Muhammad Usman; Asghar, Kashif

    2017-01-01

    Nanomedicine application in cancer immunotherapy is currently one of the most challenging areas in cancer therapeutic intervention. Innovative solutions have been provided by nanotechnology to deliver cytotoxic agents to the cancer cells partially affecting the healthy cells of the body during the process. Nanoparticle-based drug delivery is an emerging approach to stimulate the immune responses against cancer. The inhibition of indoleamine 2,3-dioxygenase (IDO) is a pivotal area of research in cancer immunotherapy. IDO is a heme-containing immunosuppressive enzyme, which is responsible for the degradation of tryptophan while increasing the concentration of kynurenine metabolites. Various preclinical studies showed that IDO inhibition in certain diseases may result in significant therapeutic effects. Here, we provide a review of the natural and synthetic inhibitors of IDO. These inhibitors are classified according to their source, inhibitory concentrations, the chemical structure, and the mechanism of action. Tumor-targeted chemotherapy is an advanced technique and has more advantages as compared to the conventional chemotherapy. Search for more efficient and less toxic nanoparticles in conjunction with compounds to inhibit IDO is still an area of interest for several research groups worldwide, especially revealing to be an extensive and a promising area in cancer therapeutic innovations. PMID:28176942

  13. Indoleamine 2,3-dioxygenase: First evidence of expression in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Cortés, Jimena; Alvarez, Claudio; Santana, Paula; Torres, Elisa; Mercado, Luis

    2016-12-01

    The role of enzymes as active antimicrobial agents of the innate immunity in teleost fish is proposed in diverse works. Secretion of Indoleamine 2,3-dioxygenase (IDO) has been described in higher vertebrates; it degrades l-tryptophan in extracellular environments associated mainly with mucosal organs. The effect of IDO on decreasing amino acid concentration may inhibit the growth of potential pathogens. In fish the study of this molecule is still. Here we report the identification of an Onchorhyncus mykiss IDO homologue (OmIDO). IDO was cloned, sequenced, and the primary structure shows conservation of key functional sites. The constitutive expression is altered when the fish is challenged with LPS as a pathogen-associated molecular pattern (PAMPs). Up-regulation of IDO was shown preferentially in the fish's mucosal cells. In order to obtain evidence of a possible regulation mechanism, an in vitro cell model was used for to show that OmIDO is induced by rIFN. These study has identified a Indoleamine 2,3-dyoxigenase in O. mykiss will contribute to expands our knowledge of the function this protein in fish immune response. These findings allow to propose the use of OmIDO as a molecular indicator of strength of the animal's immune response and wellbeing.

  14. Indoleamine 2,3 Dioxygenase (IDO Expression and Activity in Relapsing-Remitting Multiple Sclerosis.

    Directory of Open Access Journals (Sweden)

    Roberta Mancuso

    Full Text Available Interferon gamma (IFN-γ production induces the transcription of indoleamine 2,3 dioxygenase (IDO resulting in the reduction of T-cell activation and proliferation through the depletion of tryptophan and the elicitation of Treg lymphocytes. IDO was shown to be involved in the pathogenesis of autoimmune diseases; we investigated whether changes in IDO gene expression and activity could be indicative of onset of relapse in multiple sclerosis (MS patients.IDO and interferon-γ (IFN-γ gene expression, serum IDO activity (Kynurenine/Tryptophan ratio and serum neopterin concentration--a protein released by macrophages upon IFN-γ stimulation--were measured in 51 individuals: 36 relapsing remitting (RR-MS patients (21 in acute phase--AMS, 15 in stable phase--SMS and 15 healthy controls (HC. PBMCs samples in AMS patients were collected before (BT-AMS and during glucocorticoids-based therapy (DT-AMS.IDO expression was increased and IFN-γ was decreased (p<0.001 in BT-AMS compared to SMS patients. Glucocorticoids-induced disease remission resulted in a significant reduction of IDO and IFN-γ gene expression, IDO catalytic activity (p<0.001. Serum neopterin concentration followed the same trend as IDO expression and activity.Measurement of IDO gene expression and activity in blood could be a useful marker to monitor the clinical course of RR-MS. Therapeutic interventions modulating IDO activity may be beneficial in MS.

  15. Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites

    Science.gov (United States)

    Breda, Carlo; Sathyasaikumar, Korrapati V.; Sograte Idrissi, Shama; Notarangelo, Francesca M.; Estranero, Jasper G.; Moore, Gareth G. L.; Green, Edward W.; Kyriacou, Charalambos P.; Schwarcz, Robert; Giorgini, Flaviano

    2016-01-01

    Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway—kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP—the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington’s disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer’s and Parkinson’s disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits. PMID:27114543

  16. Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice.

    Science.gov (United States)

    Liu, L; Liu, H; Mah, C; Fletcher, B S

    2009-06-01

    A serious impediment to gene and protein replacement therapy in hemophilia A is the development of inhibitors. Mechanisms responsible for inhibitor development include T-cell-dependent adaptive immune responses and the CD28-B7 signaling pathway that eventually leads to the formation of antibodies directed against factor VIII (FVIII). Indoleamine 2,3-dioxygenase (IDO) is a potent immunosuppressive enzyme that can inhibit T-cell responses and induce T-cell apoptosis by regulation of tryptophan metabolism. Kynurenine, one of the metabolites of tryptophan, has been implicated as an immune modulator. Here we hypothesize that co-delivery of the genes for FVIII and IDO can attenuate inhibitor formation. Using transposon-based gene delivery, we observed long-term therapeutic FVIII expression and significantly reduced inhibitor titers when the genes were co-delivered. Co-expression of FVIII and IDO in the liver was associated with increased plasma kynurenine levels, an inhibition of T-cell infiltration and increased apoptosis of T cells within the liver. These experiments suggest that modulation of tryptophan catabolism through IDO expression provides a novel strategy to reduce inhibitor development in hemophilia gene/protein therapy.

  17. Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

    Directory of Open Access Journals (Sweden)

    Frank Elbers

    2016-01-01

    Full Text Available Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

  18. Inhibition of Indoleamine-2,3-dioxygenase (IDO in Glioblastoma Cells by Oncolytic Herpes Simplex Virus

    Directory of Open Access Journals (Sweden)

    Bonnie Reinhart

    2012-01-01

    Full Text Available Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO is expressed in response to interferon γ (IFNγ and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.

  19. Reaction mechanism of cobalt-substituted homoprotocatechuate 2,3-dioxygenase: a QM/MM study.

    Science.gov (United States)

    Cao, Lili; Dong, Geng; Lai, Wenzhen

    2015-04-01

    The reaction mechanisms of cobalt-substituted homoprotocatechuate 2,3-dioxygenase (Co-HPCD) with electron-rich substrate homoprotocatechuate (HPCA) and electron-poor substrate 4-nitrocatechol (4NC) were investigated by quantum mechanical/molecular mechanical (QM/MM) calculations. Our results demonstrated that the Co-O2 adducts has doublet ground state with a Co(III)-O2(•-) character when 4NC was used as the substrate, in good agreement with the EPR spectroscopic experiment. The reactive oxygen species is the doublet Co(III)-O2(•-) for Co-HPCD/4NC and the quartet SQ(•↑)-Co(II)-O2(•-↓) species for Co-HPCD/HPCA, indicating that the substrate plays important roles in the dioxygen activation by Co-HPCD. B3LYP was found to overestimate the rate-limiting barriers in Co-HPCD. TPSSh predicts barriers of 21.5 versus 12.0 kcal/mol for Co-HPCD/4NC versus Co-HPCD/HPCA, which is consistent with the fact that the rate of the reaction is decreased when the substrate was changed from HPCA to 4NC.

  20. Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

    Science.gov (United States)

    Gupta, S L; Carlin, J M; Pyati, P; Dai, W; Pfefferkorn, E R; Murphy, M J

    1994-01-01

    Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts. Images PMID:8188349

  1. The immune system strikes back: cellular immune responses against indoleamine 2,3-dioxygenase.

    Science.gov (United States)

    Sørensen, Rikke Baek; Berge-Hansen, Linda; Junker, Niels; Hansen, Christina Aaen; Hadrup, Sine Reker; Schumacher, Ton N M; Svane, Inge Marie; Becker, Jürgen C; thor Straten, Per; Andersen, Mads Hald

    2009-09-07

    The enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses. The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL) from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations. IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general.

  2. On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7

    KAUST Repository

    Bruno, Mark

    2014-05-01

    Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-β-carotene to form a supposedly 9-cis-configured β-apo-10′-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the β-apo-10′-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10′-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-β-carotene. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Exposition of dermatophyte Trichophyton mentagrophytes to L-cystine induces expression and activation of cysteine dioxygenase.

    Science.gov (United States)

    Kasperova, Alena; Cahlikova, Romana; Kunert, Jiri; Sebela, Marek; Novak, Zdenek; Raska, Milan

    2014-11-01

    Cysteine dioxygenase (CDO) is involved in regulation of intracellular cysteine levels by catabolising the cysteine to sulphite and sulphate. In keratinolytic fungi, sulphite is actively excreted to reduce disulphide bridges in keratin before its enzymatic degradation. The pathogenicity role of CDO was confirmed in cysteine-hypersensitive and growth-defective ΔCdo mutant of Arthroderma benhamiae on hair and nails. We analysed the CDO expression regulation in T. mentagrophytes (anamorph of A. benhamiae) mycelia by determining the Cdo mRNA and CDO protein levels and by analysing the proportion of two molecular forms of CDO in response to l-cystine exposure. Cdo mRNA levels in mycelia lysates were detected by reverse-transcription real-time polymerase chain reaction and CDO protein by western blot using mouse CDO-specific hyperimmune serum. The Cdo mRNA level increased gradually 2.5-4.5 h after exposure of the mycelium to l-cystine. The CDO protein, detected as two bands of different mobility, appeared earlier in comparison to mRNA (1 h) and culminated after 24 h. More mobile form prevailed after 4.5 h. The comparison of the dynamics in the Cdo mRNA and CDO protein levels indicates that T. mentagrophytes responds to l-cystine by increased transcription and apparently decreased degradation of the CDO and by changing towards higher mobility molecular form, similar to previous reports describing mammalian analogue.

  4. Cysteine dioxygenase type 1 promotes adipogenesis via interaction with peroxisome proliferator-activated receptor gamma

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Peng; Chen, Yi; Ji, Ning; Lin, Yunfeng; Yuan, Quan; Ye, Ling; Chen, Qianming, E-mail: qmchen@scu.edu.cn

    2015-02-27

    Mammalian cysteine dioxygenase type 1 (CDO1) is an essential enzyme for taurine biosynthesis and the biodegradation of toxic cysteine. As previously suggested, Cdo1 may be a marker of liposarcoma progression and adipogenic differentiation, but the role of Cdo1 in adipogenesis has yet been reported. In this study, we found that the expression of Cdo1 is dramatically elevated during adipogenic differentiation of 3T3-L1 pre-adipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs). Conversely, knockdown of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation in 3T3-L1 cells and mBMSCs. Mechanistically, we found Cdo1 interacted with Pparγ in response to adipogenic stimulus. Further, depletion of Cdo1 reduced the recruitment of Pparγ to the promoters of C/EBPα and Fabp4. Collectively, our finding indicates that Cdo1 may be a co-activator of Pparγ in adipogenesis, and may contribute to the development of disease associated with excessive adipose tissue. - Highlights: • Cdo1expression is highly up-regulated during adipogenic differentiation of 3T3-L1 and mBMSCs. • Depletion of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation. • Cdo1interacts with Pparγ during adipogenesis. • Knockdown of Cdo1 inhibited Pparγ binding to the promoters of C/EBPα and Fabp4.

  5. Emerging concepts on inhibitors of indoleamine 2,3-dioxygenase in rheumatic diseases.

    Science.gov (United States)

    Filippini, P; Del Papa, N; Sambataro, D; Del Bufalo, A; Locatelli, F; Rutella, S

    2012-01-01

    The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) finely regulates both innate and adaptive immune responses through the degradation of the essential amino acid tryptophan into kynurenine and other downstream metabolites, which suppress effector T-cell function and promote the differentiation of regulatory T cells. A novel role for IDO1 as a signaling molecule and a modifier of innate inflammatory responses is now emerging. In particular, IDO1 can either support or antagonize inflammation in a context- and tissuedependent manner. Studies in experimental arthritis have unravelled a previously unappreciated role for IDO in controlling B-cell activation and autoantibody production. IDO dysregulation has been documented in patients with systemic lupus erythematosus, systemic sclerosis and Sjogren's syndrome, as well as in severe sepsis and chronic kidney disease. This article summarizes the contribution of IDO to the pathophysiology of inflammatory/autoimmune disorders, and discusses whether strategies to restore metabolic equilibrium in the kynurenine pathway might be pursued in diseases states such as rheumatoid arthritis and systemic sclerosis.

  6. Indoleamine 2,3 Dioxygenase as a Potential Therapeutic Target in Huntington's Disease.

    Science.gov (United States)

    Mazarei, Gelareh; Leavitt, Blair R

    2015-01-01

    Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington's disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington's disease and will assess this enzyme as a potential therapeutic target for HD.

  7. Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy.

    Science.gov (United States)

    Martínez-Andújar, Cristina; Ordiz, M Isabel; Huang, Zhonglian; Nonogaki, Mariko; Beachy, Roger N; Nonogaki, Hiroyuki

    2011-10-11

    Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

  8. Resveratrol intake enhances indoleamine-2,3-dioxygenase activity in humans.

    Science.gov (United States)

    Gualdoni, Guido A; Fuchs, Dietmar; Zlabinger, Gerhard J; Gostner, Johanna M

    2016-10-01

    Resveratrol is a polyphenol compound found in various nutrients that was shown to have immunomodulatory, anti-cancerogenic, and cardioprotective effects. The regulation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme in inflammatory tryptophan metabolism, has been proposed to be involved in resveratrol's biological effects. These observations, however, rely on in vitro findings and animal studies. Therefore, we assessed the impact of resveratrol on tryptophan metabolism after oral intake in humans. Healthy volunteers were orally administrated 5g resveratrol (n=8) or placebo (n=2) in a pilot study. IDO activity was determined by analyzing plasma levels of tryptophan and kynurenine. Determination of the immune activation marker neopterin was included in the analysis. Resveratrol administration significantly reduced tryptophan levels 2.5h (presveratrol administration. This is the first evidence of a modulatory effect of orally administered resveratrol on tryptophan metabolism in humans. Since IDO has been shown to play a crucial role in immunity, cancer development and regulation of vascular tone, the modulation of this enzyme might be involved in resveratrol's diverse biological effects. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

  9. Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.

    Science.gov (United States)

    Zelena, Kateryna; Krings, Ulrich; Berger, Ralf G

    2012-03-01

    Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Tissue distribution, intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase.

    Science.gov (United States)

    Neve, Søren; Aarenstrup, Lene; Tornehave, Ditte; Rahbek-Nielsen, Henrik; Corydon, Thomas Juhl; Roepstorff, Peter; Kristiansen, Karsten

    2003-01-01

    4-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme involved in tyrosine catabolism. HPD was shown to be identical to a protein named the F-antigen, exploited by immunologists because of its unique immunological properties. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. Decreased expression of HPD is often observed in association with the severe type I tyrosinemia, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. In this study we present a comprehensive analysis of tissue specific expression and intracellular localization of HPD in the rat. By combined use of in situ hybridization and immunohistochemistry we confirm previously known sites of expression in liver and kidney. In addition, we show that HPD is abundantly expressed in neurons in the cortex, cerebellum and hippocampus. By using immunoelectron microscopy and confocal laser scanning microscopy, we provide evidence that HPD contrary to earlier assumptions specifically localizes to membranes of the endoplasmic reticulum and the Golgi apparatus. Detailed mass spectrometric analyses of HPD purified from rat liver revealed N-terminal and C-terminal processing of HPD, and expression of recombinant HPD suggested that C-terminal processing enhances the enzymatic activity.

  11. Expression and post-translational modification of human 4-hydroxy-phenylpyruvate dioxygenase.

    Science.gov (United States)

    Aarenstrup, Lene; Falch, Anne Marie; Jakobsen, Kirsten K; Neve, Søren; Henriksen L, Linda Ø; Tommerup, Niels; Leffers, Henrik; Kristiansen, Karsten

    2002-01-01

    4-hydroxyphenylpyruvate dioxygenase (HPD) (EC 1.13.11.27) is a key enzyme involved in tyrosine catabolism. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. The severe type I tyrosinemia, caused by a deficiency of fumarylacetoacetate hydrolase which functions downstream of HPD in the tyrosine degradation pathway, is often associated with decreased expression of HPD, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. The HPD gene was previously mapped to the chromosomal region 12q24-->qter. In the present study high-resolution chromosome mapping localized the HPD gene to 12q24.31. DNase I footprinting, revealed that four regions of the HPD promoter were protected by rat liver nuclear proteins. Computer-assisted analyses suggested that these elements might bind Sp1/AP2, HNF4, HNF3/CREB, and C/EBP, respectively. In transient transfection experiments, the proximal 271bp of the promoter conferred basal transcriptional activation in human Chang cells. Sequences in intron 1 were able to enhance the activity of this basal promoter. Finally, vaccinia virus-based expression provided evidence that HPD is subject to phosphorylation, and furthermore, allowed mapping of the HPD protein in the human keratinocyte 2D database.

  12. Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria.

    Science.gov (United States)

    Tomoeda, K; Awata, H; Matsuura, T; Matsuda, I; Ploechl, E; Milovac, T; Boneh, A; Scott, C R; Danks, D M; Endo, F

    2000-11-01

    The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of 'hawkinsin,' may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

  13. Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols

    Energy Technology Data Exchange (ETDEWEB)

    Bartels, I.; Knackmuss, H.J.; Reineke, W.

    1984-03-01

    The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K/sub 2/) were 1.62 x 10/sup -3/ sec/sup -1/ for 3-chlorocatechol and 2.38 x 10/sup -3/ sec/sup -1/ for 3-fluorocatechol. The inhibitor constants (K/sub i/) were 23 ..mu..M for 3-chlorocatechol and 17 ..mu..M for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoci acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation. 64 references.

  14. Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase

    Science.gov (United States)

    Kuehnl, Susanne; Schroecksnadel, Sebastian; Temml, Veronika; Gostner, Johanna M.; Schennach, Harald; Schuster, Daniela; Schwaiger, Stefan; Rollinger, Judith M.; Fuchs, Dietmar; Stuppner, Hermann

    2013-01-01

    Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5 μM) and trachelogenin (IC50 of 57.4 μM) showed higher activity than matairesinol (IC50 >200 μM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anti-cancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown. PMID:23867649

  15. Inhibition of indoleamine 2,3-dioxygenase activity accelerates skin wound healing.

    Science.gov (United States)

    Ito, Hiroyasu; Ando, Tatsuya; Ogiso, Hideyuki; Arioka, Yuko; Saito, Kuniaki; Seishima, Mitsuru

    2015-06-01

    Skin wound healing is a complex process involving several stages that include inflammation, proliferation, and remodeling. In the inflammatory phase, pro-inflammatory cytokines and chemokines are induced at the wound site and, they contribute to the development of wound healing. These cytokines also induce indoleamine 2,3-dioxygenase (IDO1) activity; this is the rate-limiting and first enzyme in the l-tryptophan (TRP)-l-kynurenine (KYN) pathway. This study examined the effect of IDO1 on the process of skin wound healing. The expression of the Ido1 mRNA was enhanced after creating a wound in wild-type (WT) mice. TRP concentration was simultaneously reduced at the wound site. The rate of wound healing in IDO1 knockout (IDO-KO) mice was significantly higher than that in WT mice. 1-Methyl-dl-tryptophan (1-MT), a potent inhibitor of IDO1, increased the rate of wound healing in WT mice. The administration of TRP accelerated wound healing in vivo and in an in vitro experimental model, whereas the rate of wound healing was not affected by the administration of KYN. The present study identifies the role of IDO1 in skin wound healing, and indicates that the local administration of 1-MT or TRP may provide an effective strategy for accelerating wound healing.

  16. Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression.

    Science.gov (United States)

    Kuan, Yu-Diao; Lee, Che-Hsin

    2016-01-05

    Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments.

  17. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

    Science.gov (United States)

    Rocaboy-Faquet, Emilie; Noguer, Thierry; Romdhane, Sana; Bertrand, Cédric; Dayan, Franck Emmanuel; Barthelmebs, Lise

    2014-08-01

    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.

  18. Expression Profile of Carotenoid Cleavage Dioxygenase Genes in Summer Squash (Cucurbita pepo L.).

    Science.gov (United States)

    González-Verdejo, Clara I; Obrero, Ángeles; Román, Belén; Gómez, Pedro

    2015-06-01

    Carotenoids are important dietary components that can be found in vegetable crops. The accumulation of these compounds in fruit and vegetables is altered by the activity of carotenoid cleavage dioxygenases (CCDs) enzymes that produce their degradation. The aim of this work was to study the possible implication of CCD genes in preventing carotenoid storage in the horticultural crop summer squash (Cucurbita pepo L.). The relationship between the presence of these compounds and gene expression for CCDs was studied in three varieties showing different peel and flesh colour. Expression analysis for the CCD genes CpNCED1, CpNCED2, CpNCED3, CpNCED9, CpCCD1, CpCCD4a, CpCCD4b and CpCCD8 was carried out on different organs and at several fruit developmental stages. The results showed that the CpCCD4a and CpCCD4b genes were highly expressed in the variety with lowest carotenoid content suggesting a putative role in carotenoid accumulation pattern in summer squash fruit.

  19. Indoleamine 2, 3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines

    Institute of Scientific and Technical Information of China (English)

    Shih Ling HWANG; Nancy Pei-Yee CHUNG; Jacqueline Kwai-Yi CHAN; Chen-Lung Steve LIN

    2005-01-01

    Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.

  20. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    Energy Technology Data Exchange (ETDEWEB)

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  1. Chemical origins of isoform selectivity in histone deacetylase inhibitors.

    Science.gov (United States)

    Butler, Kyle V; Kozikowski, Alan P

    2008-01-01

    Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.

  2. Isoform-targeted regulation of cardiac adenylyl cyclase.

    Science.gov (United States)

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  3. p53 isoform profiling in glioblastoma and injured brain.

    Science.gov (United States)

    Takahashi, R; Giannini, C; Sarkaria, J N; Schroeder, M; Rogers, J; Mastroeni, D; Scrable, H

    2013-06-27

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10 to 70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, some of which can modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry and reverse transcription-PCR. We found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared with tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions.

  4. Laminin isoforms in endothelial and perivascular basement membranes.

    Science.gov (United States)

    Yousif, Lema F; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.

  5. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

    Directory of Open Access Journals (Sweden)

    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  6. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  7. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  8. Acetaminophen inhibits liver trytophan-2,3-dioxygenase activity with a concomitant rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic acid.

    Science.gov (United States)

    Daya, S; Anoopkumar-Dukie, S

    2000-06-08

    The effect of the analgesic agent, acetaminophen was determined on rat forebrain serotonin levels as well as hepatic tryptophan-2,3-dioxygenase (TDO) activity and urinary 5-hydroxyindole acetic acid (5-HIAA). The results show that acetaminophen administration (100mg/kg) over three hours does not affect the holoenzyme of tryptophan-2,3-dioxygenase but significantly inhibits the apoenzyme. This inhibition is accompanied by a concomitant rise in forebrain serotonin levels. This phenomenon is also accompanied by a reduction in urinary 5-HIAA levels. These results suggest that acetaminophen use is accompanied by changes in brain serotonin levels due to inhibition of hepatic tryptophan-2,3-dioxygenase activity. This in turn could explain the possible abuse potential of acetaminophen and its effects on mood at high doses.

  9. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Fumio; Awata, Hisataka; Matsuda, Ichiro [Kumamoto Univ. (Japan)

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this train together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  10. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

    Science.gov (United States)

    Endo, F; Awata, H; Katoh, H; Matsuda, I

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  11. Resonance Raman study on indoleamine 2,3-dioxygenase: Control of reactivity by substrate-binding

    Energy Technology Data Exchange (ETDEWEB)

    Yanagisawa, Sachiko; Hara, Masayuki [Graduate School of Life Science and Picobiology Institute, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Sugimoto, Hiroshi; Shiro, Yoshitsugu [Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Koto 1-1-1, Sayo-cho, Sayo-gun, Hyogo 679-5198 (Japan); Ogura, Takashi, E-mail: ogura@sci.u-hyogo.ac.jp [Graduate School of Life Science and Picobiology Institute, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan)

    2013-06-20

    Highlights: • Indoleamine 2,3-dioygenase has been studied by resonance Raman spectroscopy. • Trp-binding to the enzyme induces high frequency shift of the Fe–His stretching mode. • Increased imidazolate character of histidine promotes the O–O bond cleavage step. • A fine-tuning of the reactivity of the O–O bond cleavage reaction is identified. • The results are consistent with the sequential oxygen-atom-transfer mechanism. - Abstract: Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron–histidine stretching mode by 3 cm{sup −1}, indicating an increase in negativity of the imidazole ring, which favors the O–O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron–histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O–O bond cleavage by the oxygenated heme upon binding of L-Trp.

  12. The immune system strikes back: cellular immune responses against indoleamine 2,3-dioxygenase.

    Directory of Open Access Journals (Sweden)

    Rikke Baek Sørensen

    Full Text Available BACKGROUND: The enzyme indoleamine 2,3-dioxygenase (IDO exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses. METHODS AND FINDINGS: The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations. CONCLUSION: IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general.

  13. Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

    Science.gov (United States)

    Wright, Terry R.; Shan, Guomin; Walsh, Terence A.; Lira, Justin M.; Cui, Cory; Song, Ping; Zhuang, Meibao; Arnold, Nicole L.; Lin, Gaofeng; Yau, Kerrm; Russell, Sean M.; Cicchillo, Robert M.; Peterson, Mark A.; Simpson, David M.; Zhou, Ning; Ponsamuel, Jayakumar; Zhang, Zhanyuan

    2010-01-01

    Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops. PMID:21059954

  14. Discovery of a Novel Linoleate Dioxygenase of Fusarium oxysporum and Linoleate Diol Synthase of Colletotrichum graminicola.

    Science.gov (United States)

    Sooman, Linda; Oliw, Ernst H

    2015-12-01

    Fungal pathogens constitute serious threats for many forms of life. The pathogenic fungi Fusarium and Colletotrichum and their formae speciales (f. spp.) infect many types of crops with severe consequences and Fusarium oxysporum can also induce keratitis and allergic conditions in humans. These fungi code for homologues of dioxygenase-cytochrome P450 (DOX-CYP) fusion proteins of the animal heme peroxidase (cyclooxygenase) superfamily. The objective was to characterize the enzymatic activities of the DOX-CYP homologue of Colletotrichum graminicola (EFQ34869) and the DOX homologue of F. oxysporum (EGU79548). The former oxidized oleic and linoleic acids in analogy with 7,8-linoleate diol synthases (LDSs), but with the additional biosynthesis of 8,11-dihydroxylinoleic acid. The latter metabolized fatty acids to hydroperoxides with broad substrate specificity. It oxidized 20:4n-6 and 18:2n-6 to hydroperoxides with an R configuration at the (n-10) positions, and other n-6 fatty acids in the same way. [11S-(2)H]18:2n-6 was oxidized with retention and [11R-(2)H]18:2n-6 with loss of deuterium, suggesting suprafacial hydrogen abstraction and oxygen insertion. Fatty acids of the n-3 series were oxidized less efficiently and often to hydroperoxides with an R configuration at both (n-10) and (n-7) positions. The enzyme spans 1426 amino acids with about 825 residues in the N-terminal domain with DOX homology and 600 residues at the C-terminal domain without homology to other enzymes. We conclude that fungal oxylipins can be formed by two novel subfamilies of cyclooxygenase-related DOX.

  15. The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase.

    Science.gov (United States)

    Huang, Chih-Wei; Liu, Hsiu-Chen; Shen, Chia-Pei; Chen, Yi-Tong; Lee, Sung-Jai; Lloyd, Matthew D; Lee, Hwei-Jen

    2016-05-01

    4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a non-haem iron(II)-dependent oxygenase that catalyses the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate (HG). In the active site, a strictly conserved 2-His-1-Glu facial triad co-ordinates the iron ready for catalysis. Substitution of these residues resulted in about a 10-fold decrease in the metal binding affinity, as measured by isothermal titration calorimetry, and a large reduction in enzyme catalytic efficiencies. The present study revealed the vital role of the ligand Glu(349) in enzyme function. Replacing this residue with alanine resulted in loss of activity. The E349G variant retained 5% activity for the coupled reaction, suggesting that co-ordinating water may be able to support activation of the trans-bound dioxygen upon substrate binding. The reaction catalysed by the H183A variant was fully uncoupled. H183A variant catalytic activity resulted in protein cleavage between Ile(267) and Ala(268) and the production of an N-terminal fragment. The H266A variant was able to produce 4-hydroxyphenylacetate (HPA), demonstrating that decarboxylation had occurred but that there was no subsequent product formation. Structural modelling of the variant enzyme with bound dioxygen revealed the rearrangement of the co-ordination environment and the dynamic behaviour of bound dioxygen in the H266A and H183A variants respectively. These models suggest that the residues regulate the geometry of the reactive oxygen intermediate during the oxidation reaction. The mutagenesis and structural simulation studies demonstrate the critical and unique role of each ligand in the function of HPPD, and which correlates with their respective co-ordination position.

  16. In vivo correction with recombinant adenovirus of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in strain III mice.

    Science.gov (United States)

    Kubo, S; Kiwaki, K; Awata, H; Katoh, H; Kanegae, Y; Saito, I; Yamamoto, T; Miyazaki, J; Matsuda, I; Endo, F

    1997-01-01

    Tyrosinemia type 3, caused by a genetic deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (HPD) in tyrosine catabolism, is characterized by convulsion, ataxia, and mental retardation. The III mouse is a model of tyrosinemia type 3. HPD activity and protein are defective in the liver and its blood tyrosine levels are elevated, the range being between 1,100 and 1,656 microM. We constructed a recombinant adenoviral vector bearing the human HPD cDNA (AdexCAGhHPD), which is expressed under the control of a potent CAG promoter. III mice were injected with 1.0 x 10(8) to 1.0 x 10(9) pfu of AdexCAGhHPD through the tail vein. When 3.0 x 10(8) - 1.0 x 10(9) pfu were injected, blood tyrosine levels decreased within 3 hr, reached a normal range (under 300 microM), and remained at a low level for 2-6 weeks. Hepatic HPD activities also increased as early as 3 hr after the injection of 5.0 x 10(8) pfu, reached the levels comparable to the control mice in 3-7 days, and then decreased, and correlated well to blood tyrosine. Hepatic HPD expression was confirmed by Northern blot and immunoblot analyses. Histology revealed no difference (gross or microscopic) between the liver injected with AdexCAGhHPD and the control. No significant changes in blood tyrosine levels were noted after the second injection of 5.0 x 10(8) pfu of AdexCAGhHPD. Thus, the intravenous administration of the adenoviral vector bearing a foreign gene seems suitable for transient, early gene transfer into the liver.

  17. Structure of 4-hydrophenylpyruvic acid dioxygenase (HPD) gene and its mutation in tyrosinemic mouse strain III

    Energy Technology Data Exchange (ETDEWEB)

    Awata, H.; Endo, F.; Matsuda, I. [Kumamoto Univ. Medical School (Japan)] [and others

    1994-09-01

    4-Hydroxphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and is developmentally regulated in mammals. A genetic deficiency of the enzyme in man and mouse leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human and mouse gene libraries. The human HPD gene is over 30 kilo-bases long and is split into 14 exons. Analysis of the 5{prime} flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5{prime} flanking region of the gene are similar to those of other genes which are specifically expressed in hepatocytes and which are developmentally regulated. The gene for mouse HPD has a similar structure and we obtained evidence for a nucleotide substitution which generates a termination codon in exon 7 of the HPD gene in III mice. This mutation associates a partial exon skipping and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Thus, mouse strain III can serve as a genetic model for human tyrosinemia type 3. Ongoing studies are expected to elucidate the disease process involved in hereditary tyrosinemia type 1 and to shed light on mechanisms that mediate developmental regulation of HPD gene expression. In addition, mouse strain III together with recently established models for tyrosinemia type 1 will facilitate studies on hereditary tyrosinemias.

  18. Characteristics and function of sulfur dioxygenase in Echiuran worm Urechis unicinctus.

    Directory of Open Access Journals (Sweden)

    Litao Zhang

    Full Text Available BACKGROUND: Sulfide is a common toxin to animals and is abundant in coastal and aquatic sediments. Sulfur dioxygenase (SDO is thought to be the key enzyme involved in sulfide oxidation in some organisms. The echiuran worm, Urechis unicinctus, inhabits coastal sediment and tolerates high concentrations of sulfide. The SDO is presumably important for sulfide tolerance in U. unicinctus. RESULTS: The full-length cDNA of SDO from the echiuran worm U. unicinctus, proven to be located in the mitochondria, was cloned and the analysis of its sequence suggests that it belongs to the metallo-β-lactamase superfamily. The enzyme was produced using an E. coli expression system and the measured activity is approximately 0.80 U mg protein(-1. Furthermore, the expression of four sub-segments of the U. unicinctus SDO was accomplished leading to preliminary identification of functional domains of the enzyme. The identification of the conserved metal I (H113, H115, H169 and D188, metal II (D117, H118, H169 and H229 as well as the potential glutathione (GSH (R197, Y231, M279 and I283 binding sites was determined by enzyme activity and GSH affinity measurements. The key residues responsible for SDO activity were identified by analysis of simultaneous mutations of residues D117 and H118 located close to the metal II binding site. CONCLUSION: The recombinant SDO from U. unicinctus was produced, purified and characterized. The metal binding sites in the SDO were identified and Y231 recognized as the mostly important amino acid residue for GSH binding. Our results show that SDO is located in the mitochondria where it plays an important role in sulfide detoxification of U. unicinctus.

  19. Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

    Directory of Open Access Journals (Sweden)

    Ferrero Marcela A

    2008-03-01

    Full Text Available Abstract Background Polycyclic aromatic hydrocarbons (PAHs, widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination. Results Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus. Conclusion These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

  20. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    OpenAIRE

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homo...

  1. Purification and properties of protocatechuate 3,4-dioxygenase from Chaetomium piluliferum induced with p-hydroxybenzoic acid.

    Science.gov (United States)

    Wojtaś-Wasilewska, M; Trojanowski, J

    1980-01-01

    1. Protocatechuate 3,4-dioxygenase (protocatechuate : oxygen 3,4-oxidoreductase, EC 1.13.11.3) was isolated from mycelium of Chaetomium piluliferum induced with p-hydroxybenzoic acid. The enzyme was purified about 80-fold by ammonium sulphate fractionation and DEAE-cellulose and Sephadex G-200 chromatography, and was homogeneous on polyacrylamide-gel electrophoresis. 2. The enzyme showed high substrate specificity; its pH optimum was 7.5-8.0, and molecula weight about 76 000 as determined by filtration on Sephadex G-200. The Michaelis constant for protocatechuic acid was 11.1 microM.

  2. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Science.gov (United States)

    Eksi, Ridvan; Li, Hong-Dong; Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S; Kretzler, Matthias; Guan, Yuanfang

    2013-01-01

    Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

  3. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Directory of Open Access Journals (Sweden)

    Ridvan Eksi

    Full Text Available Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

  4. ApoE isoform-dependent changes in hippocampal synaptic function

    Directory of Open Access Journals (Sweden)

    Sullivan Patrick M

    2009-05-01

    Full Text Available Abstract The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.

  5. Conformational Flexibility Differentiates Naturally Occurring Bet v 1 Isoforms.

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E; Ahammer, Linda; Kamenik, Anna S; Liedl, Klaus R; Tollinger, Martin

    2017-06-03

    The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform's higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

  6. Protein tyrosine phosphatase PTPRR isoforms in cellular signaling and trafficking

    NARCIS (Netherlands)

    Dilaver, Gönül

    2005-01-01

    Previous work has revealed the existence of two Protein Tyrosine Phosphatases in mouse, PTPBR7 and PTP-SL, that were in part identical, suggesting that they originated from the same gene, termed Ptprr (1,5,6). In this thesis, I report on the characterization of the various PTPRR isoforms in neuronal

  7. Murine Sirt3 protein isoforms have variable half-lives

    Science.gov (United States)

    Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3...

  8. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sial

  9. Differential water permeability and regulation of three aquaporin 4 isoforms

    DEFF Research Database (Denmark)

    Fenton, Robert A.; Moeller, Hanne B; Zelenina, Marina

    2010-01-01

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the b...

  10. Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

    Directory of Open Access Journals (Sweden)

    Apiruck Watthanasurorot

    2011-06-01

    Full Text Available The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam, encodes 9(Ig-4(FNIII-(Ig-2(FNIII-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

  11. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  12. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  13. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing;

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  14. Mast cells express novel functional IL-15 receptor alpha isoforms.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  15. Growth hormone isoforms in a girl with gigantism.

    Science.gov (United States)

    Ng, L L; Chasalow, F I; Escobar, O; Blethen, S L

    1999-01-01

    Several previous investigations have suggested that there may be different growth hormone isoforms in patients with acromegaly. We used three different site-specific monoclonal antibodies (MAbs) to investigate growth hormone (GH) isoforms in serum from an 8 year-old girl with a GH and prolactin secreting adenoma. The pattern of GH-immunoreactivity was dependent on the circumstances of collection. Serum obtained after oral glucose had very little cross reactivity with MAb 352 although concentrations of up to 15 micrograms/l were found with two other MAbs, 033 and 665. MAb 352 does not recognize the 20,000 dalton isoform of GH (20K) while both MAb 033 and 665 do. The same pattern of GH immunoreactivity (low MAb 352, equal and higher MAb 033 and 665) was seen in other baseline samples. In contrast, samples obtained after TRH/GnRH showed immunoreactivity patterns expected for a mixture of 22,000 dalton isoform of GH (22K) with only a small amount of 20K. GH samples obtained during sleep showed both patterns with episodic peaks with equal immunoreactivity superimposed on the basal pattern (decreased activity with MAb 352). Affinity chromatography of basal samples showed that a portion of the GH immunoreactivity was neither 22K nor 20K, although in stimulated samples, over 70% of GH was 22K or 20K GH. In conclusion, the nature of GH isoforms present in serum varies with GH concentration. These differences may contribute to the known difficulty in correlating disease activity and random GH measurements in patients with GH secreting adenomas.

  16. Cloning and mutagenesis of catechol 2,3-dioxygenase gene from the gram-positive Planococcus sp. strain S5.

    Science.gov (United States)

    Hupert-Kocurek, Katarzyna; Stawicka, Agnieszka; Wojcieszyńska, Danuta; Guzik, Urszula

    2013-01-01

    In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzyme's catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its Km was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.

  17. Multistep conversion of para-substituted phenols by phenol hydroxylase and 2,3-dihydroxybiphenyl 1,2-dioxygenase.

    Science.gov (United States)

    Qu, Yuanyuan; Shi, Shengnan; Ma, Qiao; Kong, Chunlei; Zhou, Hao; Zhang, Xuwang; Zhou, Jiti

    2013-04-01

    A multistep conversion system of para-substituted phenols by recombinant phenol hydroxylase (PH(IND)) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC(LA-4)) was constructed in this study. Docking studies with different para-substituted phenols and corresponding catechols inside of the active site of PH(IND) and BphC(LA-4) predicted that all the substrates should be transformed. High-performance liquid chromatography-mass spectrometry analysis showed that the products of multistep conversion were the corresponding para-substituted catechols and semialdehydes. For the first-step conversion, the formation rate of 4-fluorocatechol (0.39 μM/min/mg dry weight) by strain PH(IND) hydroxylation was 1.15, 6.50, 3.00, and 1.18-fold higher than the formation of 4-chlorocatechol, 4-bromocatechol, 4-nitrocatechol, and 4-methylcatechol, respectively. For the second-step conversion, the formation rates of semialdehydes by strain BphC(LA-4) were as follows: 5-fluoro-HODA>5-chloro-HODA>2-hydroxy-5-nitro-ODA>5-bromo-HODA>2-hydroxy-5-methyl-ODA. The present study suggested that the multistep conversion by both ring hydroxylase and cleavage dioxygenase should be potential in the synthesis of industrial precursors and provide a novel avenue in the wastewater recycling treatment.

  18. Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

    Science.gov (United States)

    Sallmann, Madleen; Kumar, Suresh; Chernev, Petko; Nehrkorn, Joscha; Schnegg, Alexander; Kumar, Devesh; Dau, Holger; Limberg, Christian; de Visser, Sam P

    2015-05-11

    Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.

  19. Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

    Science.gov (United States)

    Ilg, Andrea; Yu, Qiuju; Schaub, Patrick; Beyer, Peter; Al-Babili, Salim

    2010-08-01

    Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

  20. Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase

    Science.gov (United States)

    Lewis-Ballester, Ariel; Forouhar, Farhad; Kim, Sung-Mi; Lew, Scott; Wang, YongQiang; Karkashon, Shay; Seetharaman, Jayaraman; Batabyal, Dipanwita; Chiang, Bing-Yu; Hussain, Munif; Correia, Maria Almira; Yeh, Syun-Ru; Tong, Liang

    2016-01-01

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases. PMID:27762317

  1. Activity of a Carboxyl-Terminal Truncated Form of Catechol 2,3-Dioxygenase from Planococcus sp. S5

    Directory of Open Access Journals (Sweden)

    Katarzyna Hupert-Kocurek

    2014-01-01

    Full Text Available Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2 are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35°C, it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its Km (66.17 µM was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P<0.05 increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas.

  2. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors

    DEFF Research Database (Denmark)

    Khan, N.; Jeffers, M.; Kumar, S.;

    2008-01-01

    ) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rh...

  3. Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

    NARCIS (Netherlands)

    Kooij, V.; Venkatraman, V.; Kirk, J.A.; Ubaida-Mohien, C.; Graham, D.R.; Faber, M.J.; Eyk, J.E. Van

    2014-01-01

    PURPOSE: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. EXPER

  4. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  5. The paradoxical patterns of expression of indoleamine 2,3-dioxygenase in colon cancer

    Directory of Open Access Journals (Sweden)

    Ding Ya

    2009-08-01

    Full Text Available Abstract Background One of the putative mechanisms of tumor immune escape is based on the hypothesis that carcinomas actively create an immunosuppressed state via the expression of indoleamine 2,3-dioxygenase (IDO, both in the cancer cells and in the immune cells among the tumor-draining lymph nodes (TDLN. In an attempt to verify this hypothesis, the patterns of expression of IDO in the cancer cells and the immune cells among colon cancers were examined. Methods Seventy-one cases of pathologically-confirmed colon cancer tissues matched with adjacent non-cancerous tissues, lymph node metastases, and TDLN without metastases were collected at the Sun Yat-sen Cancer Center between January 2000 and December 2000. The expression of IDO and Bin1, an IDO regulator, was determined with an immunohistochemical assay. The association between IDO or Bin1 expression and TNM stages and the 5-year survival rate in colon cancer patients was analyzed. Results IDO and Bin1 were detected in the cytoplasm of cancer cells and normal epithelium. In primary colon cancer, the strong expression of IDO existed in 9/71 cases (12.7%, while the strong expression of Bin1 existed in 33/71 cases (46.5%. However, similar staining of IDO and Bin1 existed in the adjacent non-cancerous tissues. Among the 41 cases with primary colon tumor and lymph node metastases, decreased expression of IDO was documented in the lymph node metastases. Furthermore, among the TDLN without metastases, a higher density of IDO+cells was documented in 21/60 cases (35%. Both univariate and multivariate analyses revealed that the density of IDO+cells in TDLN was an independent prognostic factor. The patients with a higher density of IDO+cells in TDLN had a lower 5-year survival rate (37.5% than the cells with a lower density (73.1%. Conclusion This study demonstrated paradoxical patterns of expression of IDO in colon cancer. The high density IDO+cells existed in TDLN and IDO was down-regulated in lymph

  6. NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O₂ reactivity.

    Science.gov (United States)

    Hayden, Joshua A; Farquhar, Erik R; Que, Lawrence; Lipscomb, John D; Hendrich, Michael P

    2013-10-01

    Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires Fe(II) for catalysis, but Mn(II) can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient M(III)-O2 (·-) species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the M(III)-O2 (·-) species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of Fe(II) and Mn(II). Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to

  7. NO Binding to Mn-Substituted Homoprotocatechuate 2,3-Dioxygenase: Relationship to O2 Reactivity

    Science.gov (United States)

    Hayden, Joshua A.; Farquhar, Erik R.; Que, Lawrence; Lipscomb, John D.; Hendrich, Michael P.

    2014-01-01

    Homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of 3,4-dihydroxyphenylacetic acid (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron(s) from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2•− species not observed during turnover of the wild type FeHPCD. The factors governing formation of the MIII-O2•− species are explored here with EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from ability of the enzyme to maintain the ~0.8 V difference in the solution redox potentials of FeII and MnII. Due to the higher potential of Mn, the formation of the NO or O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active site His200. The same non-optimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover

  8. Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

    Directory of Open Access Journals (Sweden)

    Malihe-Sadat Poormasjedi-Meibod

    Full Text Available Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO-expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57

  9. The effects of trace elements, cations, and environmental conditions on protocatechuate 3,4-dioxygenase activity

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    Andréa Scaramal da Silva

    2013-04-01

    Full Text Available Phenanthracene is a highly toxic organic compound capable of contaminating water and soils, and biodegradation is an important tool for remediating polluted environments. This study aimed to evaluate the effects of trace elements, cations, and environmental conditions on the activity of the protocatechol 3,4-dioxygenase (P3,4O enzyme produced by the isolate Leifsonia sp. in cell-free and immobilized extracts. The isolate was grown in Luria Bertani broth medium (LB amended with 250 mg L-1 of phenanthrene. Various levels of pH (4.0-9.0, temperature (5-80 °C, time (0-90 min, trace elements (Cu2+, Hg2+ and Fe3+, and cations (Mg2+, Mn2+, K+ and NH4+ were tested to determine which conditions optimized enzyme activity. In general, the immobilized extract exhibited higher enzyme activity than the cell-free extract in the presence of trace elements and cations. Adding iron yielded the highest relative activity for both cell-free and immobilized extracts, with values of 16 and 99 %, respectively. Copper also increased enzyme activity for both cell-free and immobilized extracts, with values of 8 and 44 %, respectively. Enzyme activity in the phosphate buffer was high across a wide range of pH, reaching 80 % in the pH range between 6.5 and 8.0. The optimum temperatures for enzyme activity differed for cell-free and immobilized extracts, with maximum enzyme activity observed at 35 ºC for the cell-free extract and at 55 ºC for the immobilized extract. The cell-free extract of the P3,4O enzyme exhibited high activity only during the first 3 min of incubation, when it showed 50 % relative activity, and dropped to 0 % after 60 min of incubation. By contrast, activity in the immobilized extract was maintained during 90 min of incubation. This isolate has important characteristics for phenanthrene biodegradation, producing high quantities of the P3,4O enzyme that forms part of the most important pathway for PAH biodegradation.

  10. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Science.gov (United States)

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

  11. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Directory of Open Access Journals (Sweden)

    Angélique Guillaudeau

    Full Text Available The EGFR (epidermal growth factor receptor is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a, normal and tumor cells produce soluble EGFR isoforms (sEGFR that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2, 3 (v3 and 4 (v4 mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab and intracellular domain targeted antibody (ICD-Ab. EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade, histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS. PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

  12. The tomato CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis

    NARCIS (Netherlands)

    Kohlen, W.; Charnikhova, T.; Lammers, M.; Pollina, T.; Toth, P.; Haider, I.; Pozo, M.J.; Maagd, de R.A.; Ruyter-Spira, C.P.; Bouwmeester, H.J.; Lopez-Raez, J.A.

    2012-01-01

    •Strigolactones are plant hormones that regulate both above- and belowground plant architecture. Strigolactones were initially identified as rhizosphere signaling molecules. In the present work, the tomato (Solanum lycopersicum) CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) was cloned and its role in

  13. BW A4C and other hydroxamic acids are potent inhibitors of linoleic acid 8R-dioxygenase of the fungus Gaeumannomyces graminis.

    Science.gov (United States)

    Brodowsky, I D; Hamberg, M; Oliw, E H

    1994-03-11

    Linoleic acid is converted to 8R-hydroperoxylinoleic acid by the soluble 8R-dioxygenase of the fungus Gaeumannomyces graminis. Effects of different lipoxygenase inhibitors on the 8R-dioxygenase were evaluated. Three hydroxamic acid derivatives were investigated. BW A4C (N-(3-phenoxycinnamyl)acetohydroxamic acid) was the most potent with an IC50 of 0.2 microM, followed by zileuton (3-10 microM) and linoleate-hydroxamic acid (0.02 mM). Two other lipoxygenase inhibitors, nordihydroguaiaretic acid and eicosatetraynoic acid, were less potent (IC50 0.09 and 0.15 mM, respectively). The 8R-dioxygenase was also strongly inhibited by commonly used buffer additives, dithiothreitol, beta-mercaptoethanol and phenylmethanesulfonyl fluoride. G. graminis also contains a hydroperoxide isomerase, which converts 8R-hydroperoxylinoleic acid to 7S,8S-dihydroxylinoleic acid. Ammonium sulphate precipitation and gel filtration indicated that the dioxygenase and the hydroperoxide isomerase activities could be separated.

  14. Characterization of MnpC, a hydroquinone dioxygenase likely involved in the meta-nitrophenol degradation by Cupriavidus necator JMP134.

    Science.gov (United States)

    Yin, Ying; Zhou, Ning-Yi

    2010-11-01

    Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as the sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that MnpC might have played an important role in MNP degradation. In order to experimentally confirm the prediction, we have now characterized the mnpC-encoded (amino)hydroquinone dioxygenase involved in the ring-cleavage reaction of MNP degradation. Real-time PCR analysis indicated that mnpC played an essential role in MNP degradation. MnpC was purified to homogeneity as an N-terminal six-His-tagged fusion protein, and it was proved to be a dimer as demonstrated by gel filtration. MnpC was a Fe(2+)- and Mn(2+)-dependent dioxygenase, catalyzing the ring-cleavage of hydroquinone to 4-hydroxymuconic semialdehyde in vitro and proposed as an aminohydroquinone dioxygenase involved in MNP degradation in vivo. Phylogenetic analysis suggested that MnpC diverged from the other (chloro)hydroquinone dioxygenases at an earlier point, which might result in the preference for its physiological substrate.

  15. Modeling the 2-His-1-Carboxylate Facial Triad: Iron-Catecholato Complexes as Structural and Functional Models of the Extradiol Cleaving Dioxygenases

    NARCIS (Netherlands)

    Bruijnincx, P.C.A.; Lutz, M.; Spek, A.L.; Hagen, W.R.; Weckhuysen, B.M.; van Koten, G.; Klein Gebbink, R.J.M.

    2007-01-01

    Mononuclear iron(II)- and iron(III)-catecholato complexes with three members of a new 3,3-bis(1-alkylimidazol-2-yl)propionate ligand family have been synthesized as models of the active sites of the extradiol cleaving catechol dioxygenases. These enzymes are part of the superfamily of dioxygen-activ

  16. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    Science.gov (United States)

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  17. Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data

    DEFF Research Database (Denmark)

    Tay, Aidan P; Pang, Chi Nam Ignatius; Twine, Natalie a;

    2015-01-01

    data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against...... the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1...

  18. Characterization of four hemocyanin isoforms in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    XU Jingxiang; RUAN Lingwei; LI Zhen; YU Xiaoman; LI Sedong; SHI Hong; XU Xun

    2015-01-01

    In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in inver-tebrate.

  19. Disulfide isoforms of recombinant glia maturation factor beta.

    Science.gov (United States)

    Zaheer, A; Lim, R

    1990-09-14

    Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.

  20. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel

    2011-01-01

    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  1. Functional differences between L- and T-plastin isoforms.

    Science.gov (United States)

    Arpin, M; Friederich, E; Algrain, M; Vernel, F; Louvard, D

    1994-12-01

    Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin, C.-S., R. H. Aebersold, and J. Leavitt. 1990. Mol. Cell. Biol. 10: 1818-1821). To learn more about the biological significance of their tissue specificity, we have overproduced the T- and L-plastin isoforms in a fibroblast-like cell line, CV-1, and in a polarized epithelial cell line, LLC-PK1. In CV-1 cells, overproduction of T- and L-plastins induces cell rounding and a concomitant reorganization of actin stress fibers into geodesic structures. L-plastin remains associated with microfilaments while T-plastin is almost completely extracted after treatment of the cells with non-ionic detergent. In LLC-PK1 cells, T-plastin induces shape changes in microvilli and remains associated with microvillar actin filaments after detergent extraction while L-plastin has no effect on these structures and is completely extracted. The effect of T-plastin on the organization of microvilli differs from that of villin, another actin-bundling protein. Our experiments indicate that these two isoforms play differing roles in actin filament organization, and do so in a cell type-specific fashion. Thus it is likely that these plastin isoforms play fundamentally different roles in cell function.

  2. [Clinical relevance of myosin isoforms in the diaphragm].

    Science.gov (United States)

    Gayan-Ramirez, G; Decramer, M

    2000-06-01

    The diaphragm as a striated muscle is characterized by the repetition of a single element arranged in series: the sarcomere containing two kinds of myofilaments: a thick one constituted by the myosin, and a thin one primarily composed of actin. The myosin molecule consists of two heads where two myosin heavy chains (MHC) are fixed, a flexible hinge with two light (MLC) chains, and long rod-shaped tails. The diaphragm contains 4 MHC isoforms (MHC-slow, MHC-2A, MHC-2B, MHC-2X) and 6 MLC isoforms (MLC-1f, MLC-3f, MLC-1sa, MLC-1sb, MLC-2f, MLC-2s/v). In humans, the diaphragm contains mainly fibers expressing the isoforms MHC-slow, MHC-2A, and MLC-2f, MLC-2s et MLC-1f. For the mechanical properties of the different isoforms, there is a gradient from the MHC-slow to the MHC-2A, MHC-2B and MHC-2X/2B. According to the circumstances, the diaphragm will adapt towards a slow profile (COPD, cardiac failure and in animals: Duchenne muscular dystrophy, denervation-1 week, age-female, corticosteroids, chronic stimulation), or a fast profile (in animals: chronic hypoxia, denervation-2 weeks, age-males) or a more oxidative profile (in animals: cachexia, obesity). The reasons why the diaphragm adapts towards a slower or a faster muscle are not known. In fact, for a given pathological situation, several factors are able to influence the fiber composition of the diaphragm. Therefore, the net result of the influence of these different factors in terms of MHC and MLC diaphragm adaptation is difficult to predict.

  3. Characterization of the Human LPIN1-encoded Phosphatidate Phosphatase Isoforms*

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M.

    2010-01-01

    The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218). In this work, we characterized human lipin 1 α, β, and γ isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the α, β, and γ isoforms were dependent on Mg2+ or Mn2+ ions at pH 7.5 at 37 °C. The activities were inhibited by concentrations of Mg2+ and Mn2+ above their optimums and by Ca2+, Zn2+, N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 °C. The α, β, and γ activities followed saturation kinetics with respect to the molar concentration of PA (Km values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number ∼2) kinetics with respect to the surface concentration of PA (Km values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (kcat) for the α, β, and γ isoforms were 68.8 ± 3.5, 42.8 ± 2.5, and 5.7 ± 0.2 s−1, respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity. PMID:20231281

  4. Regulation of NADPH oxidase 5 by protein kinase C isoforms.

    Directory of Open Access Journals (Sweden)

    Feng Chen

    Full Text Available NADPH oxidase5 (Nox5 is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular

  5. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    M. R. Aquino-Silva

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  6. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    Aquino-Silva M. R.

    2003-01-01

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  7. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  8. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  9. Differential expression of syndecan isoforms during mouse incisor amelogenesis.

    Science.gov (United States)

    Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

    2007-08-01

    Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

  10. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  11. Ascorbate as a co-factor for fe- and 2-oxoglutarate dependent dioxygenases: physiological activity in tumor growth and progression.

    Science.gov (United States)

    Kuiper, Caroline; Vissers, Margreet C M

    2014-01-01

    Ascorbate is a specific co-factor for a large family of enzymes known as the Fe- and 2-oxoglutarate-dependent dioxygenases. These enzymes are found throughout biology and catalyze the addition of a hydroxyl group to various substrates. The proline hydroxylase that is involved in collagen maturation is well known, but in recent times many new enzymes and functions have been uncovered, including those involved in epigenetic control and hypoxia-inducible factor (HIF) regulation. These discoveries have provided crucial mechanistic insights into how ascorbate may affect tumor biology. In particular, there is growing evidence that HIF-1-dependent tumor progression may be inhibited by increasing tumor ascorbate levels. However, rigorous clinical intervention studies are lacking. This review will explore the physiological role of ascorbate as an enzyme co-factor and how this mechanism relates to cancer biology and treatment. The use of ascorbate in cancer should be informed by clinical studies based on such mechanistic hypotheses.

  12. Inflammation-induced activation of the indoleamine 2,3-dioxygenase pathway: Relevance to cancer-related fatigue.

    Science.gov (United States)

    Kim, Sangmi; Miller, Brian J; Stefanek, Michael E; Miller, Andrew H

    2015-07-01

    Cancer-related fatigue (CRF) is a common complication of cancer and its treatment that can significantly impair quality of life. Although the specific mechanisms remain poorly understood, inflammation is now considered to be a distinct component of CRF in addition to effects of depression, anxiety, insomnia, and other factors. One key biological pathway that may link inflammation and CRF is indoleamine 2,3-dioxygenase (IDO). Induced by inflammatory stimuli, IDO catabolizes tryptophan to kynurenine (KYN), which is subsequently converted into neuroactive metabolites. Here we summarize current knowledge concerning the relevance of the IDO pathway to CRF, including activation of the IDO pathway in cancer patients and, as a consequence, accumulation of neurotoxic KYN metabolites and depletion of serotonin in the brain. Because IDO inhibitors are already being evaluated as therapeutic agents in cancer, the elucidation of the relationship between IDO activation and CRF in cancer patients may lead to novel diagnostic and clinical approaches to managing CRF and its debilitating consequences.

  13. Characteristics and biotechnology applications of aliphatic amino acid hydroxylases belonging to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily.

    Science.gov (United States)

    Hibi, Makoto; Ogawa, Jun

    2014-05-01

    The asymmetric hydroxylation of inactive carbon atoms is still an important reaction in the industrial synthesis of valuable chiral compounds such as pharmaceuticals and fine chemicals. Applications of monooxygenation enzymes, like cytochrome P450 monooxygenases, flavin-containing monooxygenases, and Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe/αKG-DOs), are strongly desired as hydroxylation biocatalysts because they have great advantages in regio- and stereoselectivity of the reactions. Recently, several novel Fe/αKG-DOs have been found to catalyze the asymmetric hydroxylation of aliphatic amino acids. Depending on their amino acid sequences, these Fe/αKG-DOs catalyze different types of regioselective hydroxylations, or C3-, C4-, and C5-hydroxylation. Additionally, most also have stereoselective sulfoxidation activities. Here, we have reviewed the characterization and process development of this novel functioning group of Fe/αKG-DOs.

  14. Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

    Directory of Open Access Journals (Sweden)

    A.S. Silva

    2013-01-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAH are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L-1 of anthracene (PAH. The optimum conditions of pH (4.0-9.0, temperature (5-70 ºC, reaction time (10-90 min and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 ºC, 20 ºC higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract.

  15. Characterization of the fungal gibberellin desaturase as a 2-oxoglutarate-dependent dioxygenase and its utilization for enhancing plant growth.

    Science.gov (United States)

    Bhattacharya, Anjanabha; Kourmpetli, Sofia; Ward, Dennis A; Thomas, Stephen G; Gong, Fan; Powers, Stephen J; Carrera, Esther; Taylor, Benjamin; de Caceres Gonzalez, Francisco Nuñez; Tudzynski, Bettina; Phillips, Andrew L; Davey, Michael R; Hedden, Peter

    2012-10-01

    The biosynthesis of gibberellic acid (GA(3)) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA(4) desaturase (DES), is unknown. DES converts GA(4) to GA(7) by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA(3) in the growing shoots than in controls, although GA(1) content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA(3) being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.

  16. Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-ionone release.

    Science.gov (United States)

    Rubio, Angela; Rambla, José Luís; Santaella, Marcella; Gómez, M Dolores; Orzaez, Diego; Granell, Antonio; Gómez-Gómez, Lourdes

    2008-09-05

    Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound beta-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of beta-carotene and emission of beta-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.

  17. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8

    Energy Technology Data Exchange (ETDEWEB)

    Burkin, D.J.; Jones, C. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (United States)); Kimbro, K.S.; Taylor, M.W. (Indiana Univ., Bloomington, IN (United States)); Barr, B.L.; Gupta, S.L. (Hipple Cancer Research Center, Dayton, OH (United States))

    1993-07-01

    Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the catabolic pathway for tryptophan. This extrahepatic enzyme differs from the hepatic enzyme, tryptophan 2,3-dioxygenase (TDO), in molecular as well as enzymatic characteristics, although both enzymes catalyze the same reaction: cleavage of tryptophan into N-formylkynurenine. The induction of IDO by IFN-[gamma] plays a role in the antigrowth effect of IFN-[gamma] in cell cultures and in the inhibition of intracellular pathogens, e.g., Toxoplasma gondii and Chlamydia psittaci. Tryptophan is also the precursor for the synthesis of serotonin, and reduced levels of tryptophan and serotonin found in AIDS patients have been correlated with the presence of IFN-[gamma] and consequent elevation of IDO activity. The IDO enzyme has been purified and characterized, and its cDNA and genomic DNA clones have been isolated and analyzed. DNA from hybrid cells containing fragments of human chromosome 8 was used to determine the regional localization of the IDO gene on chromosome 8. The hybrids R30-5B and R30-2A contain 8p11 [yields] qter and 8q13 [yields] qter, respectively. Hybrid 229-3A contains the 8pter [yields] q11. The hybrid R30-2A was negative for the IDO gene, whereas R30-5B and 229-3A were positive as analyzed by PCR and verified by Southern blotting. Only the region close to the centromere is shared by R30-5B and 229-3A hybrids. The results indicate that the IDO gene is located on chromosome 8p11 [yields] q11.

  18. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes

    Science.gov (United States)

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  19. PSA Isoforms' Velocities for Early Diagnosis of Prostate Cancer.

    Science.gov (United States)

    Heidegger, Isabel; Klocker, Helmut; Pichler, Renate; Horninger, Wolfgang; Bektic, Jasmin

    2015-06-01

    Free prostate-specific antigen (fPSA) and its molecular isoforms are suggested for enhancement of PSA testing in prostate cancer (PCa). In the present study we evaluated whether PSA isoforms' velocities might serve as a tool to improve early PCa diagnosis. Our study population included 381 men who had undergone at least one ultrasound-guided prostate biopsy whose pathologic examination yielded PCa or showed no evidence of prostatic malignancy. Serial PSA, fPSA, and proPSA measurements were performed on serum samples covering 7 years prior to biopsy using Beckmann Coulter Access immunoassays. Afterwards, velocities of PSA (PSAV), fPSA% (fPSA%V), proPSA% (proPSA%V) and the ratio proPSA/PSA/V were calculated and their ability to discriminate cancer from benign disease was evaluated. Among 381 men included in the study, 202 (53%) were diagnosed with PCa and underwent radical prostatectomy at our Department. PSAV, fPSA%V, proPSA%V as well as proPSA/PSA/V were able to differentiate significantly between PCa and non-cancerous prostate. The highest discriminatory power between cancer and benign disease has been observed two and one year prior to diagnosis with all measured parameters. Among all measured parameters, fPSA%V showed the best cancer specificity of 45.3% with 90% of sensitivity. In summary, our results highlight the value of PSA isoforms' velocity for early detection of PCa. Especially fPSA%V should be used in the clinical setting to increase cancer detection specificity.

  20. Identification of an alternative splicing isoform of chicken Lmbr1.

    Science.gov (United States)

    Huang, Yanqun; Chen, Wen; Li, Ning; Deng, Xuemei; Kang, Xiangtao; Liu, Xiaojun

    2011-10-01

    Lmbr1 is the key candidate gene for limb development. Until now, at least five and four alternative splicing isoforms of Lmbr1 gene have been found in human and mouse, respectively. However, only two alternative splicing isoforms of this homologous gene have been reported in chicken. In the present study, one novel chicken Lmbr1 transcript variant (designated Lmbr1-1) was identified by 5' RACE and RT-PCR. Chicken Lmbr1-1 possesses one novel transcription start site different from Lmbr1-N, and was predicted to encode one 192 amino acid protein with length variation in comparison with chicken LMBR1-N protein, which was produced by 5' spliced site variation of chicken Lmbr1-N exon 10. Comparing with Lmbr1-N transcript, chicken Lmbr1-1 exhibited restricted tissue distribution of the expression. Comparative sequence analysis revealed a highly conservative intron element between chicken and mammalians from the intron 9 of chicken Lmbr1-N, indicating their possible importance as intronic elements in the regulation of alternative splicing of Lmbr1 in vertebrates. By direct PCR sequencing the exon 10 and its flanking sequences in chicken Lmbr1-N, four variation sites/haplotypes were identified from six chicken breeds. One 797A/G nonsynonymous mutation (266Arg/Gln) locating in exon 10 of chicken Lmbr1-N was predicted to affect the exon splice enhancer motif for serine/arginine-rich protein recognition. These data demonstrated that chicken Lmbr1 was alternatively spliced to generate multiple splice forms, as was the case in mammals and each of the alternative splicing isoforms might function differentially.

  1. The role of wild type RAS isoforms in cancer.

    Science.gov (United States)

    Zhou, Bingying; Der, Channing J; Cox, Adrienne D

    2016-10-01

    Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.

  2. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Directory of Open Access Journals (Sweden)

    Marina Ilicic

    2017-01-01

    Full Text Available Background. Regulation of myometrial progesterone receptor (PR expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture.

  3. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Science.gov (United States)

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  4. PKC isoforms interact with and phosphorylate DNMT1

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    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  5. The FU gene and its possible protein isoforms

    Directory of Open Access Journals (Sweden)

    Nöthen Markus M

    2004-07-01

    Full Text Available Abstract Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci. Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1 maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing

  6. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

    Directory of Open Access Journals (Sweden)

    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  7. Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy.

    Science.gov (United States)

    Iwatsubo, Kousaku; Tsunematsu, Takashi; Ishikawa, Yoshihiro

    2003-06-01

    Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoform dependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.

  8. Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

    Science.gov (United States)

    Pedrosa, M M; Legaz, M E

    1995-04-01

    Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

  9. CD44 variant isoforms in non-Hodgkin's lymphoma : a new independent prognostic factor

    OpenAIRE

    Stauder, R.; Eisterer, W.; Thaler, J; Günthert, U

    1995-01-01

    Isoforms of the transmembrane glycoprotein CD44, generated by alternative RNA splicing, have been correlated to tumor dissemination. For evaluation of the potential role of CD44 variant isoforms in non-Hodgkin's lymphoma (NHL), the presence of CD44 isoforms was analyzed in a large panel of reactive and neoplastic lymphoid tissues by immunohistochemical staining, as well as detection of CD44 variant RNAs by the reverse transcriptase-polymerase chain reaction. Whereas the CD44 standard or hemat...

  10. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  11. Amyloid-beta Isoform Metabolism Quantitation by Stable Isotope Labeled Kinetics

    OpenAIRE

    Mawuenyega, Kwasi G.; Kasten, Tom; Sigurdson, Wendy; Bateman, Randall J.

    2013-01-01

    Abundant evidence suggests a central role for the amyloid-β (Aβ) peptide in Alzheimer’s disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate th...

  12. The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

    Science.gov (United States)

    2013-10-01

    specific N-terminal peptides. Despite the high sequence homology , the isoforms show differences in cellular distribution, in localization to nuclear...Interestingly, despite the sequence homology between the isoforms and the commonality of the NLS, the isoforms show differences in their nucleus to cytoplasm...Goeres J, Sixt KM, Bekes M, Zhang XD, Salvesen GS, Matunis MJ (2009). Protection from isopeptidase-mediated deconjugation regulates paralog -selective

  13. Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang,Y.; Kang, S.; Mukherjee, T.; Bale, S.; Crane, B.; Begley, T.; Ealick, S.

    2007-01-01

    The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 {angstrom}. TDO catalyzes the irreversible oxidation of L-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate L-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.

  14. FtmOx1, a non-heme Fe(II) and alpha-ketoglutarate-dependent dioxygenase, catalyses the endoperoxide formation of verruculogen in Aspergillus fumigatus.

    Science.gov (United States)

    Steffan, Nicola; Grundmann, Alexander; Afiyatullov, Shamil; Ruan, Hanli; Li, Shu-Ming

    2009-10-07

    Verruculogen is a tremorgenic mycotoxin and contains an endoperoxide bond. In this study, we describe the cloning, overexpression and purification of a non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase FtmOx1 from Aspergillus fumigatus, which catalyses the conversion of fumitremorgin B to verruculogen by inserting an endoperoxide bond between two prenyl moieties. Incubation with (18)O(2)-enriched atmosphere demonstrated that both oxygen atoms of the endoperoxide bond are derived from one molecule of O(2). FtmOx1 is the first endoperoxide-forming non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase reported so far. A mechanism of FtmOx1-catalysed verruculogen formation is postulated and discussed.

  15. Nuclear progesterone receptor isoforms and their functions in the female reproductive tract.

    Science.gov (United States)

    Rekawiecki, R; Kowalik, M K; Kotwica, J

    2011-01-01

    Progesterone (P4), which is produced by the corpus luteum (CL), creates proper conditions for the embryo implantation, its development, and ensures proper conditions for the duration of pregnancy. Besides the non-genomic activity of P4 on target cells, its main physiological effect is caused through genomic action by the progesterone nuclear receptor (PGR). This nuclear progesterone receptor occurs in two specific isoforms, PGRA and PGRB. PGRA isoform acts as an inhibitor of transcriptional action of PGRB. The inactive receptor is connected with chaperone proteins and attachment of P4 causes disconnection of chaperones and unveiling of DNA binding domain (DBD). After receptor dimerization in the cells' nucleus and interaction with hormone response element (HRE), the receptor coactivators are connected and transcription is initiated. The ratio of these isoforms changes during the estrous cycle and reflects the different levels of P4 effect on the reproductive system. Both isoforms, PGRA and PGRB, also show a different response to the P4 receptor antagonist activity. Connection of the antagonist to PGRA can block PGRB, but acting through the PGRB isoform, P4 receptor antagonist may undergo conversion to a strongly receptor agonist. A third isoform, PGRC, has also been revealed. This isoform is the shortest and does not have transcriptional activity. Alternative splicing and insertion of additional exons may lead to the formation of different PGR isoforms. This paper summarizes the available data on the progesterone receptor isoforms and its regulatory action within the female reproductive system.

  16. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.

    Science.gov (United States)

    Schwab, Ryan S; Ihnatovych, Ivanna; Yunus, Sharifah Z S A; Domaradzki, Tera; Hofmann, Wilma A

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms.

  17. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    Science.gov (United States)

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  18. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  19. Expression of aquaporin isoforms during human and mouse tooth development.

    Science.gov (United States)

    Felszeghy, S; Módis, L; Németh, P; Nagy, G; Zelles, T; Agre, P; Laurikkala, J; Fejerskov, O; Thesleff, I; Nielsen, S

    2004-04-01

    Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

  20. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    Directory of Open Access Journals (Sweden)

    Senthil kumar M

    2011-05-01

    Full Text Available Abstract The Metallothionein (MT is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems. Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios. The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

  1. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  2. BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

    Science.gov (United States)

    Foltran, Rocío Beatriz; Diaz, Silvina Laura

    2016-07-01

    The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors. © 2016 International Society for Neurochemistry.

  3. Dual effects of indoleamine 2,3-dioxygenase inhibitors on the therapeutic effects of cyclophosphamide and cycloplatam on Ehrlich ascites tumor in mice.

    Science.gov (United States)

    Bogdanova, L A; Morozkova, T S; Amitina, S A; Mazhukin, D G; Nikolin, V P; Popova, N A; Kaledin, V I

    2014-08-01

    Ethyl pyruvate, an inhibitor of indoleamine 2,3-dioxygenase, slightly suppressed the growth of transplantable Ehrlich tumor in mice and significantly potentiated the therapeutic effect of cyclophosphamide. Another inhibitor amidoxime produced a similar effect. However, both ethyl pyruvate and amidoxime significantly reduced the effect of cycloplatam therapy. The observed changes can be stipulated by different effects of cyclophosphamide and cycloplatam on the subpopulations of lymphoid cells taking part in the formation of antitumor immunity and resistance to tumors.

  4. Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses

    OpenAIRE

    2016-01-01

    Background Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. Methods Mice in which IDO ...

  5. The mitochondrial sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 is required for amino acid catabolism during carbohydrate starvation and embryo development in Arabidopsis.

    Science.gov (United States)

    Krüßel, Lena; Junemann, Johannes; Wirtz, Markus; Birke, Hannah; Thornton, Jeremy D; Browning, Luke W; Poschet, Gernot; Hell, Rüdiger; Balk, Janneke; Braun, Hans-Peter; Hildebrandt, Tatjana M

    2014-05-01

    The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine.

  6. Analysis of multi-domain hypothetical proteins containing iron-sulphur clusters and fad ligands reveal rieske dioxygenase activity suggesting their plausible roles in bioremediation.

    Science.gov (United States)

    Sathyanarayanan, Nitish; Nagendra, Holenarasipur Gundurao

    2012-01-01

    'Conserved hypothetical' proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a 'complete' understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 40 proteins with N-terminus 2Fe-2S domain and C-terminus FNR domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicating their plausible roles in degradation of aromatic moieties.

  7. A murine model for type III tyrosinemia: lack of immunologically detectable 4-hydroxyphenylpyruvic acid dioxygenase enzyme protein in a novel mouse strain with hypertyrosinemia.

    Science.gov (United States)

    Endo, F; Katoh, H; Yamamoto, S; Matsuda, I

    1991-04-01

    We have characterized a new mutant strain of mouse that has hypertyrosinemia. The blood tyrosine level was persistently high, and increased amounts of 4-hydroxyphenylpyruvic acid and its derivatives were excreted into the urine. Succinylacetone was not detected in urine samples from these mice. All the animals were apparently healthy, and there was no evidence of hepatorenal dysfunction. The hypertyrosinemia was transmitted through an autosomal recessive inheritance. Analyses of hepatic enzymes related to tyrosine metabolism revealed that 4-hydroxyphenylpyruvic acid dioxygenase activity was virtually absent, while fumarylacetoacetase and tyrosine aminotransferases (cytosolic and mitochondrial forms) were normal in these mutant mice. Immunoblot analysis of 4-hydroxyphenylpyruvic acid dioxygenase protein in the liver indicated that the subunit protein of the enzyme was absent. It would appear that hypertyrosinemia in this mutant strain was caused by a genetic defect in 4-hydroxyphenylpyruvic acid dioxygenase. These features are similar to type III tyrosinemia in humans. Analysis of this mutant strain of mouse is expected to provide valuable information on the pathogenesis of human type III tyrosinemia and can also serve as a useful system for studies on tyrosine metabolism.

  8. The Mitochondrial Sulfur Dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 Is Required for Amino Acid Catabolism during Carbohydrate Starvation and Embryo Development in Arabidopsis1[C][W

    Science.gov (United States)

    Krüßel, Lena; Junemann, Johannes; Wirtz, Markus; Birke, Hannah; Thornton, Jeremy D.; Browning, Luke W.; Poschet, Gernot; Hell, Rüdiger; Balk, Janneke; Braun, Hans-Peter; Hildebrandt, Tatjana M.

    2014-01-01

    The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine. PMID:24692429

  9. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides.

    Science.gov (United States)

    Silver, Kristopher S; Soderlund, David M

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel alpha subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na(v)1.2a, Na(v)1.4, Na(v)1.5, and Na(v)1.8 sodium channel alpha subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat beta1 auxiliary subunit on the sensitivity of the Na(v)1.2a and Na(v)1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel alpha subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na(v)1.4 > Na(v)1.2a > Na(v)1.5 > Na(v)1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na(v)1.8 isoform was most sensitive, the Na(v)1.4 isoform was completely insensitive, and the sensitivities of the Na(v)1.5 and Na(v)1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na(v)1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na(v)1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na(v)1.2a or Na(v)1.4 isoforms with the beta1 subunit was the modest reduction in the sensitivity of the Na(v)1.2a isoform to RH 3421. These results demonstrate that mammalian sodium channel isoforms differ in their sensitivities to pyrazoline-type insecticides.

  10. Nesprins: tissue-specific expression of epsilon and other short isoforms.

    Directory of Open Access Journals (Sweden)

    Nguyen Thuy Duong

    Full Text Available Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These "muscle-specific" isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and

  11. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  12. Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer

    Science.gov (United States)

    Hjortsø, Mads Duus; Larsen, Stine Kiaer; Kongsted, Per; Met, Özcan; Frøsig, Thomas Mørch; Andersen, Gitte Holmen; Ahmad, Shamaila Munir; Svane, Inge Marie; Becker, Jürgen C; Straten, Per thor; Andersen, Mads Hald

    2015-01-01

    Tryptophan-2,3-dioxygenase (TDO) physiologically regulates systemic tryptophan levels in the liver. However, numerous studies have linked cancer with activation of local and systemic tryptophan metabolism. Indeed, similar to other heme dioxygenases TDO is constitutively expressed in many cancers. In the present study, we detected the presence of both CD8+ and CD4+ T-cell reactivity toward TDO in peripheral blood of patients with malignant melanoma (MM) or breast cancer (BC) as well as healthy subjects. However, TDO-reactive CD4+ T cells constituted distinct functional phenotypes in health and disease. In healthy subjects these cells predominately comprised interferon (IFN)γ and tumor necrosis factor (TNF)-α producing Th1 cells, while in cancer patients TDO-reactive CD4+ T-cells were more differentiated with release of not only IFNγ and TNFα, but also interleukin (IL)-17 and IL-10 in response to TDO-derived MHC-class II restricted peptides. Hence, in healthy donors (HD) a Th1 helper response was predominant, whereas in cancer patients CD4+ T-cell responses were skewed toward a regulatory T cell (Treg) response. Furthermore, MM patients hosting a TDO-specific IL-17 response showed a trend toward an improved overall survival (OS) compared to MM patients with IL-10 producing, TDO-reactive CD4+ T cells. For further characterization, we isolated and expanded both CD8+ and CD4+ TDO-reactive T cells in vitro. TDO-reactive CD8+ T cells were able to kill HLA-matched tumor cells of different origin. Interestingly, the processed and presented TDO-derived epitopes varied between different cancer cells. With respect to CD4+ TDO-reactive T cells, in vitro expanded T-cell cultures comprised a Th1 and/or a Treg phenotype. In summary, our data demonstrate that the immune modulating enzyme TDO is a target for CD8+ and CD4+ T cell responses both in healthy subjects as well as patients with cancer; notably, however, the functional phenotype of these T-cell responses differ

  13. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    Science.gov (United States)

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  14. Insulin receptor isoforms : an integrated view focused on gestational diabetes mellitus

    NARCIS (Netherlands)

    Westermeier, F.; Sáez, T.; Arroyo, P.; Toledo, F.; Gutierrez, J.; Sanhueza, C.; Pardo, F.; Leiva, A.; Sobrevia, L.

    2016-01-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms r

  15. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease.

    Science.gov (United States)

    Kamphuis, Willem; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-03-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Roles of the troponin isoforms during indirect flight muscle development in Drosophila

    Indian Academy of Sciences (India)

    Salam Herojeet Singh; Prabodh Kumar; Nallur B. Ramachandra; Upendra Nongthomba

    2014-08-01

    Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

  17. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

  18. Roles of the troponin isoforms during indirect flight muscle development in Drosophila.

    Science.gov (United States)

    Singh, Salam Herojeet; Kumar, Prabodh; Ramachandra, Nallur B; Nongthomba, Upendra

    2014-08-01

    Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

  19. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

    DEFF Research Database (Denmark)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T

    2014-01-01

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms...

  20. Mitochondrial localization of the OAS1 p46 isoform associated with a common single nucleotide polymorphism

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Pahus, Jytte; Hansen, Mariann Fagernæs

    2014-01-01

    vacuoles/lysosomes. By using recombinantly expressed OAS1 mutant proteins, we found that the OAS1 SNP rs1131454 (former rs3741981) did not affect the enzymatic OAS1 activity. Conclusions: The SNP rs10774671 determines differential expression of the OAS1 isoforms. In Daudi and HT1080 cells the p46 isoform...

  1. Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

    Science.gov (United States)

    Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A; Larsson, Lars; Ge, Ying

    2016-04-01

    Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases.

  2. Translational control of C/EBPalpha and C/EBPbeta isoform expression

    NARCIS (Netherlands)

    Calkhoven, C F; Müller, C; Leutz, A

    2000-01-01

    Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms re

  3. Cloning and characterization of the neural isoforms of human dystonin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, A.; Dalpe, G.; Mathieu, M.; Kothary, R. [Univ. of Montreal, Quebec (Canada)

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.

  4. Differences in expression, actions and cocaine regulation of two isoforms for the brain transcriptional regulator NAC1.

    Science.gov (United States)

    Korutla, L; Wang, P J; Lewis, D M; Neustadter, J H; Stromberg, M F; Mackler, S A

    2002-01-01

    BTB/POZ proteins can influence the cell cycle and contribute to oncogenesis. Many family members are present in the mammalian CNS. Previous work demonstrated elevated NAC1 mRNA levels in the rat nucleus accumbens in response to cocaine. NAC1 acts like other BTB/POZ proteins that regulate transcription but is unusual because of the absence of identifiable DNA binding domains. cDNAs were isolated encoding two NAC1 isoforms differing by only 27 amino acids (the longer isoform contains 514 amino acids). The mRNAs for both isoforms were simultaneously expressed throughout the rat brain and peripheral tissues. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that the mRNA of the longer isoform was more abundant than the mRNA of the shorter isoform. Western blot analysis demonstrated a similar unequal distribution between the isoforms in the CNS. The longer isoform was the more abundant of the two NAC1 proteins and the ratio between them differed throughout the rat brain. The shorter isoform was not detected in most of the examined peripheral tissues, suggesting differences from the CNS in post-transcriptional processing. Both isoforms repressed transcription in H293T cells using a Gal4-luciferase reporter system. However, the shorter isoform did not repress transcription as effectively as the longer isoform. Transfection of different ratios for both isoforms, in order to replicate the relative amounts observed throughout the CNS, supported an interaction between the isoforms. The net effect on transcriptional repression was determined by the ratio of the two NAC1 isoforms. Each isoform exhibited the subnuclear localization that is characteristic of many BTB/POZ proteins. A rapid and transient increase in the level of the shorter isoform occurred in the nucleus accumbens 2 h following a single i.p. cocaine injection. We conclude that the two isoforms of NAC1 may differentially affect neuronal functions, including the regulation of

  5. Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

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    Llewellyn Lynda

    2007-06-01

    Full Text Available Abstract Background Flatfish metamorphosis is a thyroid hormone (TH driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT, a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. Results In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Conclusion Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

  6. Functional Cooperativity between ABCG4 and ABCG1 Isoforms.

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    Zoltán Hegyi

    Full Text Available ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system.

  7. Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

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    Iraê A. Guerrini

    2005-01-01

    Full Text Available Abscisic acid (ABA regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (https://forests.esalq.usp.br. A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED, the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g, which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process.

  8. Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution.

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    Paissé, Sandrine; Goñi-Urriza, Marisol; Stalder, Thibault; Stadler, Thibault; Budzinski, Hélène; Duran, Robert

    2012-04-01

    The early functional response of a bacterial community from the sediments of a chronically oil-polluted retention basin located at the Etang de Berre (France) was investigated just after petroleum addition. After removing hydrocarbon compounds by natural abiotic and biotic processes, the sediments were maintained in microcosms and Vic Bilh petroleum was added. The diversity and the expression of genes encoding ring-hydroxylating dioxygenases (RHD) were examined just after the petroleum addition until 14 days focussing on the first hours following the contamination. RHD gene copy numbers and diversity were maintained throughout all the incubation period; however, transcripts were detected only during the first 2 days. One dominant RHD gene, immediately and specifically expressed in response to petroleum contamination, was related to RHD gene carried by a plasmid found in Pseudomonas spp. The expression of the RHD genes was correlated with high biodegradation levels observed for low molecular weight PAHs at 7 days of incubation. The study shows that the bacterial metabolism induced just after the oil input is a key stage that could determine the bacterial community structure changes. Monitoring the expression of RHD genes, key genes involved in hydrocarbon degradation, may provide useful information for managing bioremediation processes.

  9. Dynamic changes in bacterial community structure and in naphthalene dioxygenase expression in vermicompost-amended PAH-contaminated soils.

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    Di Gennaro, Patrizia; Moreno, Beatriz; Annoni, Emanuele; García-Rodríguez, Sonia; Bestetti, Giuseppina; Benitez, Emilio

    2009-12-30

    The aim of the present study was to explore the potential for using vermicompost from olive-mill waste as an organic amendment for enhanced bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils. The focus was to analyse the genetic potential and the naphthalene dioxygenase (NDO) expression of the bacterial communities involved in the degradation of naphthalene, as chemical model for the degradation of PAH. The structure of the metabolically active bacterial population was evidenced in the RNA-based denaturing gradient gel electrophoresis (DGGE) profiles. The relative expression of NDO was determined with real-time PCR in both the soil and the vermicompost cDNA. Naphthalene changed the structure of the metabolically active bacterial community in the vermicompost when this was artificially contaminated. When used as amendment, naphthalene-free vermicompost modified the bacterial population in the PAH-contaminated soil, evidenced in the DGGE gels after 1 month of incubation. In the amended soil, the vermicompost enhanced the NDO enzyme expression with a concomitant biodegradation of naphthalene. The effect of the vermicompost was to induce the expression of biodegradation indicator genes in the autochthonous bacterial community and/or incorporate new bacterial species capable of degrading PAH. The results indicated that vermicompost from olive-mill wastes could be considered a suitable technology to be used in PAH bioremediation.

  10. Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango

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    Singh, Rajesh K.; Ali, Sharique A.; Nath, Pravendra; Sane, Vidhu A.

    2011-01-01

    Mango is characterized by high tocopherol and carotenoid content during ripening. From a cDNA screen of differentially expressing genes during mango ripening, a full-length p-hydroxyphenylpyruvate dioxygenase (MiHPPD) gene homologue was isolated that encodes a key enzyme in the biosynthesis of tocopherols. The gene encoded a 432-amino-acid protein. Transcript analysis during different stages of ripening revealed that the gene is ripening related and rapidly induced by ethylene. The increase in MiHPPD transcript accumulation was followed by an increase in tocopherol levels during ripening. The ripening-related increase in MiHPPD expression was also seen in response to abscisic acid and to alesser extent to indole-3-acetic acid. The expression of MiHPPD was not restricted to fruits but was also seen in other tissues such as leaves particularly during senescence. The strong ethylene induction of MiHPPD was also seen in young leaves indicating that ethylene induction of MiHPPD is tissue independent. Promoter analysis of MiHPPD gene in tomato discs and leaves of stable transgenic lines of Arabidopsis showed that the cis elements for ripening-related, ethylene-responsive, and senescence-related expression resided within the 1590 nt region upstream of the ATG codon. Functionality of the gene was demonstrated by the ability of the expressed protein in bacteria to convert p-hydroxyphenylpyruvate to homogentisate. These results provide the first evidence for HPPD expression during ripening of a climacteric fruit. PMID:21430290

  11. Structural characterization of Pandoraea pnomenusa B-356 biphenyl dioxygenase reveals features of potent polychlorinated biphenyl-degrading enzymes.

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    Christopher L Colbert

    Full Text Available The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356. BPDO(B356, a heterohexameric (αβ(3 Rieske oxygenase (RO, catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356 with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356 and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.

  12. Altering substrate specificity of catechol 2,3-dioxygenase from Planococcus sp. strain S5 by random mutagenesis.

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    Hupert-Kocurek, Katarzyna; Wojcieszyńska, Danuta; Guzik, Urszula

    2014-01-01

    c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.

  13. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme.

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    Ahrazem, Oussama; Rubio-Moraga, Angela; Berman, Judit; Capell, Teresa; Christou, Paul; Zhu, Changfu; Gómez-Gómez, Lourdes

    2016-01-01

    The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.

  14. Triterpenoids isolated from the rhizomes and roots of Gentiana scabra and their inhibition of indoleamine 2,3-dioxygenase.

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    Li, Wei; Li, Lin Ying; Zhou, Wei; Hwang, Inkyu; Ma, Jin Yeul; Kim, Young Ho

    2015-12-01

    Gentiana scabra Bunge (Gentianaceae) is an important traditional Chinese medicine commonly used as a stomachic or appetite stimulant. In this study, 21 triterpenoids (1-21) were isolated from a methanol extract of the rhizomes and roots of G. scabra. Their structures were elucidated by comparing spectroscopic data with reported values. Among the isolated triterpenoids, scabanol (2) was firstly isolated from natural sources. All isolated compounds were evaluated for their inhibitory activity against indoleamine 2,3-dioxygenase (IDO), which catalyzes the rate limiting reaction for the conversion of tryptophan to kynurenine. Compounds 10 and 11 showed significant inhibitory activities, with IC(50) values of 12.5 and 9.5 μM, respectively. Compound 12 showed a moderate inhibitory effect, with an IC(50) value of 18.7 μM. Compounds 2 and 13 showed weaker inhibitory effects, with IC(50) values of 56.8 and 60.6 μM, respectively. Kynurenine is a potent immune modulator to suppress the functions of a variety of immune cells including T cells and natural killer cells. Given that, our results that a few selected triterpenoids inhibit IDO warrant further studies on their effects on the host immune system as natural immune stimulators.

  15. 4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme.

    Science.gov (United States)

    Raspail, Corinne; Graindorge, Matthieu; Moreau, Yohann; Crouzy, Serge; Lefèbvre, Bertrand; Robin, Adeline Y; Dumas, Renaud; Matringe, Michel

    2011-07-22

    4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

  16. Reaction mechanism of homoprotocatechuate 2,3-dioxygenase with 4-nitrocatechol: implications for the role of substrate.

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    Dong, Geng; Lai, Wenzhen

    2014-02-20

    The reaction mechanism of the dioxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) with the substrate 4-nitrocatechol was investigated by quantum mechanical/molecular mechanical calculations. Our results demonstrated that the experimentally determined side-on iron-oxygen complex in crystallo is a semiquinone substrate radical (SQ(•))-Fe(III)-hydroperoxo species, which could not act as the reactive species. In fact, the Fe(III)-superoxo species with a hydrogen bond between His200 and the proximal oxygen is the reactive oxygen species. The second-sphere His200 residue was found to play an important role in manipulating the orientation of the superoxide in the Fe-O2 adduct for the further reaction. The rate-limiting step is the attack of the superoxo group on the substrate with a barrier of 17.2 kcal/mol, in good agreement with the experimental value of 16.8 kcal/mol. The reaction mechanism was then compared with the one for HPCD with its native substrate homoprotocatechuate studied recently by the same methods, in which a hybrid SQ(•)-Fe(II)-O2(•-)/Fe(III)-O2(•-) was suggested to be the reactive species. Therefore, our studies suggested that the substrate plays important roles in the dioxygen activation by HPCD.

  17. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger.

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    Matsuda, J; Okabe, S; Hashimoto, T; Yamada, Y

    1991-05-25

    Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger.

  18. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

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    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  19. Indoleamine 2,3-dioxygenase (IDO) is expressed at feto-placental unit throughout mouse gestation: An immunohistochemical study

    Science.gov (United States)

    Hemmati, Shayda; Jeddi-Tehrani, Mahmood; Torkabadi, Ebrahim; Ghassemi, Jamileh; Kazemi sefat, Golnaz Ensieh; Danesh, Parivash; Barzegar Yarmohammadi, Leila; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan

    2009-01-01

    Introduction The cells expressing Indoleamine 2, 3-dioxygenase (IDO) in feto-maternal interface mediate tryptophan catabolism, hence protect allogeneic fetus from lethal rejection by maternal immune responses. In this study, we report immuno-localization of IDO+ cells in murine reproductive tract and placenta throughout mouse pregnancy by immunohistochemistry. Materials and Methods Syngeneic pregnant mice were examined for vaginal plug to discover about their state of pregnancy. A total of three pregnant mice were examined at each stage.The examination was further confirmed by the detection of sperm in vaginal smear. On the gestational days of 2nd, 12th and 18th, the uterus and oviduct were removed and expression of IDO was investigated in the endometrium, placenta and oviduct by immunohistochemistry. Results Our results showed that IDO is expressed consistently in feto-maternal interface throughout pregnancy. In endometrium, expression of IDO was predominantly confined to luminal and glandular epithelial cells. Cells at junctional and labyrinth zones of placenta showed strong IDO immunoreactivity as well. Conclusion Expression of IDO at the protein level in reproductive tract of pregnant mice during entire periods of gestation points to its potential protective role in maintenance of pregnancy. In our knowledge this is the first report of expression of IDO in feto-maternal phase during murine pregnancy. PMID:23926466

  20. New insight into the cleavage reaction of Nostoc sp. strain PCC 7120 carotenoid cleavage dioxygenase in natural and nonnatural carotenoids.

    Science.gov (United States)

    Heo, Jinsol; Kim, Se Hyeuk; Lee, Pyung Cheon

    2013-06-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8'-carotenal at 3 positions, C-13 C-14, C-15 C-15', and C-13' C-14', revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4'-diaponeurosporene, 4,4'-diaponeurosporen-4'-al, 4,4'-diaponeurosporen-4'-oic acid, 4,4'-diapotorulene, and 4,4'-diapotorulen-4'-al to generate novel cleavage products (apo-14'-diaponeurosporenal, apo-13'-diaponeurosporenal, apo-10'-diaponeurosporenal, apo-14'-diapotorulenal, and apo-10'-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.

  1. Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to Aspergillus fumigatus Infections.

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    Nan Jiang

    Full Text Available Indoleamine 2,3-dioxygenase (IDO, which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs, known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO.

  2. Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707.

    Science.gov (United States)

    Seo, Jiyoung; Kang, Su-Il; Won, Dongho; Kim, Mihyang; Ryu, Ji-Young; Kang, Suk-Woo; Um, Byung-Hun; Pan, Cheol-Ho; Ahn, Joong-Hoon; Chong, Youhoon; Kanaly, Robert A; Han, Jaehong; Hur, Hor-Gil

    2011-03-01

    Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

  3. Scavenging properties of neutrophil 4-hydroxyphenylpyruvate dioxygenase are based on a hypothesis that does not stand up to scrutiny.

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    Salerno, Costantino; Zicari, Alessandra; Mari, Emanuela; D'Eufemia, Patrizia

    2014-10-01

    It was previously reported by D'Eufemia et al. [9] that neutrophil preparations from a patient with tyrosinemia type III, i.e. with inherited deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPPD), exhibited a far higher NO release than controls, when NO was estimated in terms of nitrite content in the suspending media. It was hypothesized that HPPD might participate to NO sequestration in neutrophils and that excessive NO release might reflect the lack of the scavenging action in defective cells. In recent control experiments, we found that HPPD activity in neutrophils preparations from healthy subjects is below the detection limit of the enzymatic assay (less than 3nmol product/h per mg protein). This indicates that HPPD concentration in neutrophils is very low, if any, confirming what was already suggested in literature, and rules out the possibility of a prominent role of HPPD as NO scavenger in these cells. Moreover, we found that 500μM l-tyrosine increases nitrite release and accumulation in suspending media of U-937 cells, a human monoblast-like lymphoma cell line which displays many characteristics of macrophages, including the expression of inducible and endothelial nitric oxide synthases. We hypothesize that the increase of nitrite release by patient's neutrophils might be related to the presence of high l-tyrosine concentrations in the blood samples (426μmol/L instead of 52.1±10.9μmol/L as healthy subjects), rather than to HPPD deficiency of in these cells.

  4. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    Science.gov (United States)

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  5. Unity in diversity, a systems approach to regulating plant cell physiology by 2-oxoglutarate-dependent dioxygenases.

    Science.gov (United States)

    Kundu, Siddhartha

    2015-01-01

    Could a disjoint group of enzymes synchronize their activities and execute a complex multi-step, measurable, and reproducible response? Here, I surmise that the alpha-ketoglutarate dependent superfamily of non-haem iron (II) dioxygenases could influence cell physiology as a cohesive unit, and that the broad spectra of substrates transformed is an absolute necessity to this portrayal. This eclectic group comprises members from all major taxa, and participates in pesticide breakdown, hypoxia signaling, and osmotic stress neutralization. The oxidative decarboxylation of 2-oxoglutarate to succinate is coupled with a concomitant substrate hydroxylation and, in most cases, is followed by an additional specialized conversion. The domain profile of a protein sequence was used as an index of miscellaneous reaction chemistry and interpreted alongside existent kinetic data in a linear model of integrated function. Statistical parameters were inferred by the creation of a novel, empirically motivated flat-file database of over 3800 sequences (DB2OG) with putative 2-oxoglutarate dependent activity. The collated information was categorized on the basis of existing annotation schema. The data suggests that 2OG-dependent enzymes incorporate several desirable features of a systems level player. DB2OG, is free, accessible without a login to all users, and available at the following URL (http://comp-biol.theacms.in/DB2OG.html).

  6. Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content.

    Science.gov (United States)

    Jube, Sandro L R; Borthakur, Dulal

    2010-04-01

    The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

  7. Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells.

    Science.gov (United States)

    Hara, Toshiaki; Yamakura, Fumiyuki; Takikawa, Osamu; Hiramatsu, Rie; Kawabe, Tsutomu; Isobe, Ken-Ichi; Nagase, Fumihiko

    2008-03-20

    Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway regulates T-cell responses in some dendritic cells (DC) such as plasmacytoid DC. A Kyn assay using HPLC showed that samples were frequently deproteinized with trichloroacetic acid (TCA). In the present study, bone marrow-derived myeloid DC (BMDC) were differentiated from mouse bone marrow cells with GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with NO production in BMDC cultured for 24 h. The concentrations of Kyn in the culture supernatants were not increased by stimulation with CpG but rather decreased by based on the Kyn assay after deproteinization with TCA. The level of Kyn exogenously added into the cell-free culture supernatant of BMDC stimulated with CpG was severely decreased by deproteinization with TCA but not methanol, and the decrease was prevented when BMDC was stimulated with CpG in the presence of a NOS inhibitor. Under acidic conditions, Kyn reacted with nitrite produced by BMDC, and generated a new compound that was not detected by Ehrlich reagent reacting with the aromatic amino residue of Kyn. An analysis by mass spectrometry showed the new compound to be a diazotization form of Kyn. In conclusion, the deproteinization of samples by acidic treatment should be avoided for the Kyn assay when NO is produced.

  8. Lowering intercellular melatonin levels by transgenic analysis of indoleamine 2,3-dioxygenase from rice in tomato plants.

    Science.gov (United States)

    Okazaki, Masateru; Higuchi, Kenji; Aouini, Asma; Ezura, Hiroshi

    2010-10-01

    Melatonin exists in numerous living organisms including vertebrates, insects, fungi, bacteria, and plants. Extensive studies have been conducted on the physiological roles of melatonin in various plant species. In plants, melatonin seems to act in antioxidant protection, as a growth promoter, and in photoperiodism. However, the mechanisms by which melatonin carries out these roles remain unclear. We manipulated the endogenous melatonin content in tomato plants by modifying the metabolic enzyme indoleamine 2,3-dioxygenase (IDO). The OsIDO gene was isolated from rice (Oryza sativa) and characterized using 3-D homology modeling and reverse genetic approaches. The amino acid sequence of OsIDO showed high homology to the Ustilago maydis IDO. The 3-D model structure of OsIDO is composed of a small and a large domain. Transgenic tomato plants constitutively expressing the OsIDO gene exhibited a decrease in their melatonin content. Moreover, the number of lateral leaflets decreased in transgenic plants. Protein extracts taken from these plants showed activity degradation, demonstrating the function of OsIDO. These results suggest the involvement of IDO in plant melatonin metabolism and a possible role in plant leaf development. © 2010 The Authors. Journal of Pineal Research © 2010 John Wiley & Sons A/S.

  9. Eosinophil Granulocytes Account for Indoleamine 2,3-Dioxygenase-Mediated Immune Escape in Human Non Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Simonetta Astigiano

    2005-04-01

    Full Text Available Indoleamine 2,3-dioxygenase (IDO, a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non small cell lung cancer (NSCLC. Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

  10. Molecular docking and dynamic simulation studies evidenced plausible immunotherapeutic anticancer property by Withaferin A targeting indoleamine 2,3-dioxygenase.

    Science.gov (United States)

    Reddy, S V G; Reddy, K Thammi; Kumari, V Valli; Basha, Syed Hussain

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic drug target for the treatment of cancer characterized by pathological immune suppression. IDO catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway. Reduction in local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to the immunosuppressive effects of IDO. Presence of IDO on dentritic cells in tumor-draining lymph nodes leading to the activation of T cells toward forming immunosuppressive microenvironment for the survival of tumor cells has confirmed the importance of IDO as a promising novel anticancer immunotherapy drug target. On the other hand, Withaferin A (WA) - active constituent of Withania Somnifera ayurvedic herb has shown to be having a wide range of targeted anticancer properties. In the present study conducted here is an attempt to explore the potential of WA in attenuating IDO for immunotherapeutic tumor arresting activity and to elucidate the underlying mode of action in a computational approach. Our docking and molecular dynamic simulation results predict high binding affinity of the ligand to the receptor with up to -11.51 kcal/mol of energy and 3.63 nM of IC50 value. Further, de novo molecular dynamic simulations predicted stable ligand interactions with critically important residues SER167; ARG231; LYS377, and heme moiety involved in IDO's activity. Conclusively, our results strongly suggest WA as a valuable small ligand molecule with strong binding affinity toward IDO.

  11. Molecular Modeling and Dynamic Simulation of Arabidopsis Thaliana Carotenoid Cleavage Dioxygenase Gene: A Comparison with Bixa orellana and Crocus Sativus.

    Science.gov (United States)

    Priya, R; Sneha, P; Rivera Madrid, Renata; Doss, C George Priya; Singh, Pooja; Siva, Ramamoorthy

    2017-09-01

    Carotenoid cleavage dioxygenase (CCD) gene, ubiquitously found in numerous types of plants, are eminent in synthesizing the various volatile compounds (β-ionone, C13 -norisoprenoid, geranylacetone) known as apocarotenoids. These apocarotenoids have various biological functions such as volatile signals, allelopathic interaction and plant defense. In Arabidopsis genome sequence, four potential CCD genes have been identified namely CCD1, CCD4, CCD7, and CCD8. These four genes give rise to diverse biological functions with almost similar sequence identity. In this investigation, an in silico analysis was proposed to study CCD proteins in Arabidopsis thaliana, aiming at constructing three-dimensional (3D) structure for CCD1 proteins of Bixa orellana and Crocus sativus to observe the structural difference among AtCCD (A. thaliana CCD) proteins. The quality of modeled structures was evaluated using RAMPAGE, PSVS protein validation server and Q Mean server. Finally, we utilised molecular dynamics simulation to identify the stability of the predicted CCD protein structures. The molecular dynamic simulation also revealed that AtCCD4 protein showed lesser stability when compared to other CCD proteins. Overall results from molecular dynamics analysis predicted that BoCCD1, CsCCD1, and AtCCD1 show similar structural characteristics. J. Cell. Biochem. 118: 2712-2721, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Relationship of Abortion and the Expression of Indoleamine 2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To study the relationship of abortion and the expression of indoleamine 2,3- dioxygenase (IDO) in villus and syncytiotrophoblast in vitro.Methods RT-PCR was applied to analyze the mRNA transcription of IDO in villus of normal pregnancy and inevitable abortion and JAR cells as well. Immunohistochemistry was applied to analyze the expression of IDO protein in villus. Western blot was applied to determinate the expression of IDO protein on cultured syncytiotrophoblast. Highperformance liquid chromatography was applied to determinate whether there was kynurenine in cell culture medium of syncytiotrophoblast.Results The expression of IDO mRNA and protein in villus of inevitable abortion was lower than that of normal pregnancy; IDO mRNA did not express in JAR cells. IDO protein expressed on cultured syncytiotrophoblast, and there was kynurenine in cell culture medium of syncytiotrophoblast.Conclusion Appropriate expression of IDO in villus is necessary for maintenance of normal pregnancy and an active IDO protein expresses in syncytiotrophoblast.

  13. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  14. Patchwork assembly of nag-like nitroarene dioxygenase genes and the 3-chlorocatechol degradation cluster for evolution of the 2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeri ZWLR2-1.

    Science.gov (United States)

    Liu, Hong; Wang, Shu-Jun; Zhang, Jun-Jie; Dai, Hui; Tang, Huiru; Zhou, Ning-Yi

    2011-07-01

    Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.

  15. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    Science.gov (United States)

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  16. Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

    DEFF Research Database (Denmark)

    Kjelgaard-Hansen, Mads Jens; Christensen, Michelle B.; Lee, Marcel Huisung

    2007-01-01

    with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local...... production of these isoforms in the canine inflamed joint....

  17. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation.

    Directory of Open Access Journals (Sweden)

    Ansgar Zoch

    Full Text Available The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

  18. Role of nuclear progesterone receptor isoforms in uterine pathophysiology.

    Science.gov (United States)

    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    Progesterone is a key hormonal regulator of the female reproductive system. It plays a major role to prepare the uterus for implantation and in the establishment and maintenance of pregnancy. Actions of progesterone on the uterine tissues (endometrium, myometrium and cervix) are mediated by the combined effects of two progesterone receptor (PR) isoforms, designated PR-A and PR-B. Both receptors function primarily as ligand-activated transcription factors. Progesterone action on the uterine tissues is qualitatively and quantitatively determined by the relative levels and transcriptional activities of PR-A and PR-B. The transcriptional activity of the PR isoforms is affected by specific transcriptional coregulators and by PR post-translational modifications that affect gene promoter targeting. In this context, appropriate temporal and cell-specific expression and function of PR-A and PR-B are critical for normal uterine function. Relevant studies describing the role of PRs in uterine physiology and pathology (endometriosis, uterine leiomyoma, endometrial cancer, cervical cancer and recurrent pregnancy loss) were comprehensively searched using PubMed, Cochrane Library, Web of Science, and Google Scholar and critically reviewed. Progesterone, acting through PR-A and PR-B, regulates the development and function of the endometrium and induces changes in cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs promotes myometrial relaxation and cervical closure. Withdrawal of PR-mediated progesterone signaling triggers menstruation and parturition. PR-mediated progesterone signaling is anti-mitogenic in endometrial epithelial cells, and as such, mitigates the tropic effects of estrogen on eutopic normal endometrium, and on ectopic implants in endometriosis. Similarly, ligand-activated PRs function as tumor suppressors in endometrial cancer cells through inhibition of key cellular signaling pathways

  19. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

  20. Distinct and shared transcriptomes are regulated by microphthalmia-associated transcription factor isoforms in mast cells.

    Science.gov (United States)

    Shahlaee, Amir H; Brandal, Stephanie; Lee, Youl-Nam; Jie, Chunfa; Takemoto, Clifford M

    2007-01-01

    The Microphthalmia-associated transcription factor (Mitf) is an essential basic helix-loop-helix leucine zipper transcription factor for mast cell development. Mice deficient in Mitf harbor a severe mast cell deficiency, and Mitf-mutant mast cells cultured ex vivo display a number of functional defects. Therefore, an understanding of the genetic program regulated by Mitf may provide important insights into mast cell differentiation. Multiple, distinct isoforms of Mitf have been identified in a variety of cell types; we found that Mitf-a, Mitf-e, and Mitf-mc were the major isoforms expressed in mast cells. To determine the physiologic function of Mitf in mast cells, we restored expression of these isoforms in primary mast cells from Mitf(-/-) mice. We found that these isoforms restored granular morphology and integrin-mediated migration. By microarray analysis, proteases, signaling molecules, cell surface receptor, and transporters comprised the largest groups of genes up-regulated by all isoforms. Furthermore, we found that isoforms also regulated distinct genes sets, suggesting separable biological activities. This work defines the transcriptome regulated by Mitf in mast cells and supports its role as master regulator of mast cell differentiation. Expression of multiple isoforms of this transcription factor may provide for redundancy of biological activities while also allowing diversity of function.

  1. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Science.gov (United States)

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  2. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2016-05-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

  3. Estrogen and progesterone receptor isoforms expression in the stomach of Mongolian gerbils

    Institute of Scientific and Technical Information of China (English)

    Milena Saqui-Salces; Teresa Neri-Gómez; Armando Gamboa-Dominguez; Guillermo Ruiz-Palacios; Ignacio Camacho-Arroyo

    2008-01-01

    AIM:We studied the estrogen receptor (ER) and progesterone receptor (PR) isoforms expression in gastric antrum and corpus of female gerbils and their regulation by estradiol (E2) and progesterone (P4).METHODS: Ovariectomized adult female gerbils were subcutaneously treated with E2,and E2 + P4.Uteri and stomachs were removed,the latter were cut along the greater curvature,and antrum and corpus were excised.Proteins were immunoblotted using antibodies that recognize ER-alpha,ER-beta,and PR-A and PR-B receptor isoforms.Tissues from rats treated in the same way were used as controls.RESULTS: Specific bands were detected for ER-alpha (68 KDa),and PR isoforms (85 and 120 KDa for PR-A and PR-B isoforrns,respectively) in uteri,gastric antrum and corpus.We could not detect ER-beta isoform.PR isoforms were not regulated by E2 or P4 in uterus and gastric tissues of gerbils.ER-alpha isoform content was significantly down-regulated by E2 in the corpus,but not affected by hormones in uterus and gastric antrum.CONCLUSION: The presence of ER-alpha and PR isoforms in gerbils stomach suggests that E2 and P4 actions in this organ are in part mediated by their nuclear receptors.

  4. Identification and evolutionary analysis of tissue-specific isoforms of mitochondrial complex I subunit NDUFV3.

    Science.gov (United States)

    Guerrero-Castillo, Sergio; Cabrera-Orefice, Alfredo; Huynen, Martijn A; Arnold, Susanne

    2017-03-01

    Mitochondrial complex I is the largest respiratory chain complex. Despite the enormous progress made studying its structure and function in recent years, potential regulatory roles of its accessory subunits remained largely unresolved. Complex I gene NDUFV3, which occurs in metazoa, contains an extra exon that is only present in vertebrates and thereby evolutionary even younger than the rest of the gene. Alternative splicing of this extra exon gives rise to a short NDUFV3-S and a long NDUFV3-L protein isoform. Complexome profiling revealed that the two NDUFV3 isoforms are constituents of the multi-subunit complex I. Further mass spectrometric analyses of complex I from different murine and bovine tissues showed a tissue-specific expression pattern of NDUFV3-S and NDUFV3-L. Hence, NDUFV3-S was identified as the only isoform in heart and skeletal muscle, whereas in liver, brain, and lung NDUFV3-L was expressed as the dominant isoform, together with NDUFV3-S present in all tissues analyzed. Thus, we identified NDUFV3 as the first out of 30 accessory subunits of complex I present in vertebrate- and tissue-specific isoforms. Interestingly, the tissue-specific expression pattern of NDUFV3-S and NDUFV3-L isoforms was paralleled by changes in kinetic parameters, especially the substrate affinity of complex I. This may indicate a regulatory role of the NDUFV3 isoforms in different vertebrate tissues.

  5. Revisiting the identification of canonical splice isoforms through integration of functional genomics and proteomics evidence.

    Science.gov (United States)

    Li, Hong-Dong; Menon, Rajasree; Omenn, Gilbert S; Guan, Yuanfang

    2014-12-01

    Canonical isoforms in different databases have been defined as the most prevalent, most conserved, most expressed, longest, or the one with the clearest description of domains or posttranslational modifications. In this article, we revisit these definitions of canonical isoforms based on functional genomics and proteomics evidence, focusing on mouse data. We report a novel functional relationship network-based approach for identifying the highest connected isoforms (HCIs). We show that 46% of these HCIs are not the longest transcripts. In addition, this approach revealed many genes that have more than one highly connected isoforms. Averaged across 175 RNA-seq datasets covering diverse tissues and conditions, 65% of the HCIs show higher expression levels than nonhighest connected isoforms at the transcript level. At the protein level, these HCIs highly overlap with the expressed splice variants, based on proteomic data from eight different normal tissues. These results suggest that a more confident definition of canonical isoforms can be made through integration of multiple lines of evidence, including HCIs defined by biological processes and pathways, expression prevalence at the transcript level, and relative or absolute abundance at the protein level. This integrative proteogenomics approach can successfully identify principal isoforms that are responsible for the canonical functions of genes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    Science.gov (United States)

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

  7. Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line

    Institute of Scientific and Technical Information of China (English)

    Shu-Jie XIA; Xiao-Da TANG; Qing-Zheng MA

    2001-01-01

    Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5,6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3H-DHT) to these three isoforms was inhibited by the addition ofl00-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isofonns is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.

  8. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    Science.gov (United States)

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Inulin isoforms differ by repeated additions of one crystal unit cell.

    Science.gov (United States)

    Cooper, Peter D; Barclay, Thomas G; Ginic-Markovic, Milena; Gerson, Andrea R; Petrovsky, Nikolai

    2014-03-15

    Inulin isoforms, especially delta inulin, are important biologically as immune activators and clinically as vaccine adjuvants. In exploring action mechanisms, we previously found regular increments in thermal properties of the seven-member inulin isoform series that suggested regular additions of some energetic structural unit. Because the previous isolates carried additional longer chains that masked defining ranges, these were contrasted with new isoform isolates comprising only inulin chain lengths defining that isoform. The new series began with 19 fructose units per chain (alpha-1 inulin), increasing regularly by 6 fructose units per isoform. Thus the 'energetic unit' equates to 6 fructose residues per chain. All isoforms showed indistinguishable X-ray diffraction patterns that were also identical with known inulin crystals. We conclude that an 'energetic unit' equates to one helix turn of 6 fructose units per chain as found in one unit cell of the inulin crystal. Each isoform chain comprised progressively more helix turns plus one additional fructose and glucose residues per chain.

  10. Detection of VEGF-A(xxx)b isoforms in human tissues.

    Science.gov (United States)

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  11. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells.

    Science.gov (United States)

    Müller-Ostermeyer, F; Claus, P; Grothe, C

    2001-01-01

    Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

  12. Non-muscle Myosin II Isoforms Co-assemble in Living Cells

    Science.gov (United States)

    Beach, Jordan R.; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A.

    2014-01-01

    SUMMARY Non-muscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB and IIC), each of which possesses distinct biophysical properties and supports unique, as well as redundant, cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear if NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or if filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged versions of NM IIA, IIB and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms co-assemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly-spread cells, arguing for the existence of sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NM II isoforms. PMID:24814144

  13. Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

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    Stefanie K Schweinhuber

    Full Text Available The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as

  14. Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

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    Hisayo Sadamoto

    2010-05-01

    Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

  15. The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions.

    Science.gov (United States)

    Cooper, Peter D; Barclay, Thomas G; Ginic-Markovic, Milena; Petrovsky, Nikolai

    2013-10-01

    In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous → alpha-1 (AI-1) → alpha-2 (AI-2) → gamma (GI) → delta (DI) → zeta (ZI) → epsilon (EI) → omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using (1)H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

  16. Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

    Science.gov (United States)

    Yamanaka, Hidenori; Takeyoshi, Masahiro; Minobe, Yasushi; Yakabe, Yoshikuni; Takatsuki, Mineo; Sato, Hisaya

    2007-12-01

    Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

  17. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  18. The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily*

    Science.gov (United States)

    Zhu, Xiaofeng; van Pée, Karl-Heinz; Naismith, James H.

    2010-01-01

    Pyrrolnitrin (3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-l-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both l-tryptophan or 7-Cl-l-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In l-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr321 and Ser332 play a key role in binding these groups. The structures suggest that catalysis requires an l-configured substrate. Isothermal titration calorimetry data suggest d-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications. PMID:20421301

  19. Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

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    Tartarini Stefano

    2011-01-01

    Full Text Available Abstract Background Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch., and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH and its white-fleshed mutant 'Redhaven Bianca' (RHB were examined. Results The two genotypes displayed marked differences in the accumulation of carotenoid pigments in mesocarp tissues. Lower carotenoid levels and higher levels of norisoprenoid volatiles were observed in RHB, which might be explained by differential activity of carotenoid cleavage dioxygenase (CCD enzymes. In fact, the ccd4 transcript levels were dramatically higher at late ripening stages in RHB with respect to RH. The two genotypes also showed differences in the expression patterns of several carotenoid and isoprenoid transcripts, compatible with a feed-back regulation of these transcripts. Abamine SG - an inhibitor of CCD enzymes - decreased the levels of both isoprenoid and non-isoprenoid volatiles in RHB fruits, indicating a complex regulation of volatile production. Conclusions Differential expression of ccd4 is likely to be the major determinant in the accumulation of carotenoids and carotenoid-derived volatiles in peach fruit flesh. More in general, dioxygenases appear to be key factors controlling volatile composition in peach fruit, since abamine SG-treated 'Redhaven Bianca' fruits had strongly reduced levels of norisoprenoids and other volatile classes. Comparative functional studies of peach carotenoid

  20. Respiratory Syncytial Virus-Infected Mesenchymal Stem Cells Regulate Immunity via Interferon Beta and Indoleamine-2,3-Dioxygenase

    Science.gov (United States)

    Cheung, Michael B.; Sampayo-Escobar, Viviana; Green, Ryan; Moore, Martin L.; Mohapatra, Subhra; Mohapatra, Shyam S.

    2016-01-01

    Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD. PMID:27695127

  1. High activity of indoleamine 2,3 dioxygenase enzyme predicts disease severity and case fatality in bacteremic patients.

    Science.gov (United States)

    Huttunen, Reetta; Syrjänen, Jaana; Aittoniemi, Janne; Oja, Simo S; Raitala, Annika; Laine, Janne; Pertovaara, Marja; Vuento, Risto; Huhtala, Heini; Hurme, Mikko

    2010-02-01

    Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan (trp) catabolism, may play a critical role in various inflammatory disorders. Recent studies on trauma patients have suggested that the degradation of trp is associated with the development of sepsis. The role of IDO activity in bacteremic patients is unclear. We studied IDO activity in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, beta-hemolytic streptococcae, or Eschericia coli. The serum concentrations of trp and its metabolite kynurenine (kyn) were measured by reverse-phase high-performance liquid chromatography 1 to 4 days after the positive blood culture and on recovery. The kyn-to-trp ratio (kyn/trp), reflecting the activity of the IDO enzyme, was calculated. The maximum value in the ratio for every patient during 1 to 4 days after positive blood culture was used in analysis. The maximum kyn/trp ratio was significantly higher in nonsurvivors versus those who survived (193.7 vs. 82.4 micromol/mmol; P = 0.001). The AUC(ROC) of maximal kyn/trp in the prediction of case fatality was 0.75 (95% confidence interval, 0.64-0.87), and the kyn/trp ratio at a cutoff level of 120 micromol/mmol showed 83% sensitivity and 69% specificity for fatal disease. A kyn/trp ratio greater than 120 micromol/mmol was associated with increased risk of death versus low (

  2. Indoleamine 2,3-dioxygenase 1 (IDO1 activity correlates with immune system abnormalities in multiple myeloma

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    Bonanno Giuseppina

    2012-12-01

    Full Text Available Abstract Background Multiple myeloma (MM is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1 degrades tryptophan into kynurenine (KYN, which inhibits effector T cells and promote regulatory T-cell (Treg differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment. Methods We investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3 or smoldering MM (SMM; n=4. IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF and with the frequency of Treg cells and NY-ESO-1-specific CD8+ T cells. Results KYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4+CD25+FoxP3+ Treg cells and the contraction of NY-ESO-1-specific CD8+ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4+ T cells into bona fide CD4+CD25hiFoxP3hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO+ myeloma disease compared with patients having IDO- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity. Conclusions These data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.

  3. Immunoprotective role of indoleamine 2,3-dioxygenase in engraftment of allogenic skin substitute in wound healing.

    Science.gov (United States)

    Bahar, Mohammad Ali; Nabai, Layla; Ghahary, Aziz

    2012-01-01

    Delayed wound healing can significantly impact survival of patients who suffer from severe thermal injury. In general, the use of a wound coverage, particularly with those of bilayer skin substitute, would be ideal to promote healing and prevent infection and fluid loss. Although the use of an autologous skin substitute is desirable, its preparation is time consuming and its immediate availability is impossible. To overcome this difficulty, the authors have previously demonstrated that the expression of indoleamine 2,3 dioxygenase (IDO) could function as a local immune suppressive factor in protecting allogenic fibroblasts and keratinocytes without using any immunosuppressive medication in a wound healing animal model. IDO, which is naturally expressed in the placenta by trophoblast cells during pregnancy, plays an essential role in maternal tolerance toward the fetus. The potent and selective local immunosuppressive function of IDO makes this enzyme a very promising tool for engineering a nonrejectable skin allograft. Here, the authors reviewed and discussed how the expression of IDO by the primary cells of our skin substitute can serve as a source of IDO enzyme activity and generate a tryptophan-deficient environment. Under this condition, only skin cells but not immune cells (CD4(+) and CD8(+) cells) would survive and protect engraftment of this engineered and shelf-ready skin substitute to be used not only as wound coverage but also as a rich source of wound healing promoting factors. Therefore, this review summarizes the body of work on immunoprotective role of IDO in engraftment of allogenic skin substitute in wound healing, which has recently been reported by the authors' research group and others.

  4. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    Science.gov (United States)

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

  5. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

    Science.gov (United States)

    Kovaleva, Elena G; Rogers, Melanie S; Lipscomb, John D

    2015-09-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

  6. Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1 Gene in Primary Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Naoko Minatani

    Full Text Available Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1 gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004. Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007. Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

  7. Role of 9-Lipoxygenase and α-Dioxygenase Oxylipin Pathways as Modulators of Local and Systemic Defense

    Institute of Scientific and Technical Information of China (English)

    Jorge Vicente; Tomás Cascón; Begonya Vicedo; Pilar García-Agustín; Mats Hamberg; Carmen Castresana

    2012-01-01

    Plant 9-lipoxygenases(9-LOX)and α-dioxygenases(α-DOX)initiate the synthesis of oxylipins after bacterial infection.Here,the role of these enzymes in plants' defense was investigated using individual Arabidopsis thaliana lox1 and dox1 mutants and a double lox1 dox1 mutant.Studies with Pseudomonas syringae pv.tomato(Pst)revealed the enhanced susceptibility of lox1 to the virulent strain Pst DC3000 and the partial impairment of lox1 and dox1 mutants to activate systemic acquired resistance.Notably,both defects were enhanced in the lox1 dox1 plants as compared with individual mutants.We found that pre-treatment with 9-LOX- and α-DOX-generated oxylipins protected plant tissues against bacterial infection.The strongest effect in this respect was exerted by 9-ketooctadecatrienoic acid(9-KOT),which is produced from linolenic acid by 9-LOX.Quantification of 9-KOT revealed its accumulation after bacterial infection.The levels were reduced in lox1 and lox1 dox1 plants but strongly increased in the dox1 mutant due to metabolic interaction of the two pathways.Transcriptional analyses indicated that 9-KOT pre-treatment modifies hormone homeostasis during bacterial infection.The nature of the changes detected suggested that 9-KOT interferes with the hormonal changes caused by bacterial effectors.This notion was substantiated by the finding that 9-KOT failed to reduce the growth of PstDC3000hrpA,a mutant compromised in effector secretion,and of the avirulent strain Pst DC3000 avrRpm1.Further support for the action of the 9-LOX- and α-DOX-oxylipin pathways as modulators of hormone homeostasis was the observation that lox1 dox1 seedlings are hypersensitive to the growth-inhibitory effect of ABA and showed enhanced activation of ABA-inducible marker genes as compared with wild-type plants.

  8. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants.

    Science.gov (United States)

    Lin, J; Milase, R N

    2015-12-01

    This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

  9. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    Science.gov (United States)

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  10. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA.

    Science.gov (United States)

    Hashimoto, Hideharu; Pais, June E; Dai, Nan; Corrêa, Ivan R; Zhang, Xing; Zheng, Yu; Cheng, Xiaodong

    2015-12-15

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1-5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. A novel l-isoleucine-4′-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis

    Science.gov (United States)

    Smirnov, Sergey V; Sokolov, Pavel M; Kotlyarova, Veronika A; Samsonova, Natalya N; Kodera, Tomohiro; Sugiyama, Masakazu; Torii, Takayoshi; Hibi, Makoto; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-01-01

    A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone. PMID:23554367

  12. Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

    Science.gov (United States)

    Tuan, Pham Anh; Park, Sang Un

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

  13. An ancient relative of cyclooxygenase in cyanobacteria is a linoleate 10S-dioxygenase that works in tandem with a catalase-related protein with specific 10S-hydroperoxide lyase activity.

    Science.gov (United States)

    Brash, Alan R; Niraula, Narayan P; Boeglin, William E; Mashhadi, Zahra

    2014-05-09

    In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.

  14. Differential susceptibility of RAE-1 isoforms to mouse cytomegalovirus.

    Science.gov (United States)

    Arapovic, Jurica; Lenac, Tihana; Antulov, Ronald; Polic, Bojan; Ruzsics, Zsolt; Carayannopoulos, Leonidas N; Koszinowski, Ulrich H; Krmpotic, Astrid; Jonjic, Stipan

    2009-08-01

    The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1delta, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1delta and RAE-1gamma in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1delta form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1delta compared to RAE-1gamma but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1delta. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.

  15. Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82.

    OpenAIRE

    Takizawa, N; Kaida, N; Torigoe, S; Moritani,T.; Sawada, T.; Satoh, S.; Kiyohara, H

    1994-01-01

    Naphthalene and phenanthrene are transformed by enzymes encoded by the pah gene cluster of Pseudomonas putida OUS82. The pahA and pahB genes, which encode the first and second enzymes, dioxygenase and cis-dihydrodiol dehydrogenase, respectively, were identified and sequenced. The DNA sequences showed that pahA and pahB were clustered and that pahA consisted of four cistrons, pahAa, pahAb, pahAc, and pahAd, which encode ferredoxin reductase, ferredoxin, and two subunits of the iron-sulfur prot...

  16. Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

    Science.gov (United States)

    Montero-Pardo, Arnulfo; Diaz, Daniel; Olivares, Aleida; González-Padilla, Everardo; Murcia, Clara; Gómez-Chavarín, Margarita; Gutiérrez-Ospina, Gabriel; Perera-Marín, Gerardo

    2015-12-01

    Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

  17. Detection of isoform-specific fibroblast growth factor receptors by whole-mount in situ hybridization in early chick embryos.

    Science.gov (United States)

    Nishita, Junko; Ohta, Sho; Bleyl, Steven B; Schoenwolf, Gary C

    2011-06-01

    We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms.

  18. VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues

    Science.gov (United States)

    1996-01-01

    VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle- associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells. PMID:8567721

  19. [Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Arabtseva, M A; Lavrinenko, I A; Parfenova, I V; Grechkina, M V; Abud, F S

    2011-01-01

    Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

  20. Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration

    Science.gov (United States)

    Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S.

    2016-11-01

    Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

  1. Neuronal and intestinal protein kinase d isoforms mediate Na+ (salt taste)-induced learning.

    Science.gov (United States)

    Fu, Ya; Ren, Min; Feng, Hui; Chen, Lu; Altun, Zeynep F; Rubin, Charles S

    2009-08-11

    Ubiquitously expressed protein kinase D (PKD) isoforms are poised to disseminate signals carried by diacylglycerol (DAG). However, the in vivo regulation and functions of PKDs are poorly understood. We show that the Caenorhabditis elegans gene, dkf-2, encodes not just DKF-2A, but also a second previously unknown isoform, DKF-2B. Whereas DKF-2A is present mainly in intestine, we show that DKF-2B is found in neurons. Characterization of dkf-2 null mutants and transgenic animals expressing DKF-2B, DKF-2A, or both isoforms revealed that PKDs couple DAG signals to regulation of sodium ion (Na+)-induced learning. EGL-8 (a phospholipase Cbeta4 homolog) and TPA-1 (a protein kinase Cdelta homolog) are upstream regulators of DKF-2 isoforms in vivo. Thus, pathways containing EGL-8-TPA-1-DKF-2 enable learning and behavioral plasticity by receiving, transmitting, and cooperatively integrating environmental signals targeted to both neurons and intestine.

  2. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease

    NARCIS (Netherlands)

    Kamphuis, W.; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-01-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of ast

  3. Receptor-isoform-selective insulin analogues give tissue-preferential effects

    DEFF Research Database (Denmark)

    Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi

    2011-01-01

    The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

  4. Altered β-Amyloid Precursor Protein Isoforms in Mexican Alzheimer’s Disease Patients

    Directory of Open Access Journals (Sweden)

    V. J. Sánchez-González

    2006-01-01

    Full Text Available Objective: To determine the β-amyloid precursor protein (βAPP isoforms ratio as a risk factor for Alzheimer’s Disease and to assess its relationship with demographic and genetic variables of the disease.

  5. Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy

    Science.gov (United States)

    Bae, Sun Sik; Lee, Min Young; Rhee, Harin; Kim, Il Young; Seong, Eun Young; Lee, Dong Won; Lee, Soo Bong; Kwak, Ihm Soo; Lovett, David H.

    2017-01-01

    Background We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2) in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2) and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2) generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms. Methods We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE) and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). The streptozotocin (STZ) murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study. Results Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold). Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively). Conclusions The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for

  6. Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

    OpenAIRE

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Michael H Gelb; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h...

  7. Role of ROCK Isoforms in Regulation of Stiffness Induced Myofibroblast Differentiation in Lung Fibrosis.

    Science.gov (United States)

    Htwe, Su S; Cha, Byung H; Yue, Kan; Khademhosseini, Ali; Knox, Alan J; Ghaemmaghami, Amir M

    2017-02-22

    Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

  8. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Directory of Open Access Journals (Sweden)

    Niels Jacob Aachmann-Andersen

    Full Text Available The membrane-assisted isoform immunoassay (MAIIA quantitates erythropoietin (EPO isoforms as percentages of migrated isoforms (PMI. We evaluated the effect of recombinant human EPO (rhEPO on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13; high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13; or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3 % (mean (SD. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2% (p<0.00001 and 45.2 (7.3% (p<0.00001. Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8% (p<0.00001 and 46.1 (10.4% (p<0.00001. In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4% (p=0.029; low-dose Epoetin beta: 73.1 (17.8% (p=0.039. In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  9. Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells.

    Science.gov (United States)

    Cappelletti, Vera; Miodini, Patrizia; Di Fronzo, Giovanni; Daidone, Maria Grazia

    2006-05-01

    High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

  10. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

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    Thomas P Stricker

    2017-03-01

    Full Text Available Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+ subtype and fourteen triple negative (TN subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

  11. Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk

    DEFF Research Database (Denmark)

    Jacobsen, Stine; Niewold, T.A.; Kornalijnslijper, E.

    2005-01-01

    The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis.......The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis....

  12. Differential Regulation of Human Thymosin Beta 15 Isoforms by Transforming Growth Factor Beta 1

    Science.gov (United States)

    Banyard, Jacqueline; Barrows, Courtney; Zetter, Bruce R.

    2009-01-01

    We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080 and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322 and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility. PMID:19296525

  13. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    Science.gov (United States)

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

  14. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Science.gov (United States)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p=0.029); low-dose Epoetin beta: 73.1 (17.8)% (p=0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  15. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  16. Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer

    Science.gov (United States)

    2009-02-01

    TITLE: Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer PRINCIPAL INVESTIGATOR: Jianghua Wang, M.D...6 JAN 2009 / / /4. TITLE AND SUBTITLE Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer 5a. CONTRACT NUMBER W81XWH...quantitative RT-PCR arrays we have identified candidate mediators of these phenotypic effects . We propose to extend these studies to primary prostate epithelial

  17. The impact of tropomyosins on actin filament assembly is isoform specific.

    Science.gov (United States)

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  18. Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae.

    Science.gov (United States)

    Gorman, Maureen J; Sullivan, Lucinda I; Nguyen, Thi D T; Dai, Huaien; Arakane, Yasuyuki; Dittmer, Neal T; Syed, Lateef U; Li, Jun; Hua, Duy H; Kanost, Michael R

    2012-03-01

    Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min⁻¹ mM⁻¹. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min⁻¹ mM⁻¹; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min⁻¹ mM⁻¹. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.

  19. Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

    Directory of Open Access Journals (Sweden)

    Marion Schiengold

    2006-01-01

    Full Text Available The multidrug resistance (MDR phenotype is associated with the expression of P-glycoprotein (Pgp, coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different phases of the estrous cycle. Additionally, females were ovariectomized, submitted to different hormone treatments, and their uterus was analyzed for the expression of mdr isoforms. The results show that in the adrenal gland and ovaries mdr1 is the main isoform during proestrus, and that progesterone or a combination of progesterone and estrogen induce the expression of all mdr isoforms in the uterus of ovariectomized females. We suggest that the functions of mdr1 and mdr3 are overlapping, that mdr3 may be the more efficient isoform in the detoxification function, and that mdr1 may be more closely related to the secretion of steroid hormones.

  20. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

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    Valeria Hansberg-Pastor

    2015-01-01

    Full Text Available The CCAAT/enhancer-binding protein beta (C/EBPβ is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

  1. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    Science.gov (United States)

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  2. Profiling alternatively spliced mRNA isoforms for prostate cancer classification

    Directory of Open Access Journals (Sweden)

    Fan Jian-Bing

    2006-04-01

    Full Text Available Abstract Background Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. Results As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. Conclusion These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays.

  3. Molecular modeling study on tunnel behavior in different histone deacetylase isoforms.

    Directory of Open Access Journals (Sweden)

    Sundarapandian Thangapandian

    Full Text Available Histone deacetylases (HDACs have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E in HDAC10 and leucine (L in HDAC 11 based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.

  4. Divergent roles for thyroid hormone receptor β isoforms in the endocrine axis and auditory system

    Science.gov (United States)

    Abel, E. Dale; Boers, Mary-Ellen; Pazos-Moura, Carmen; Moura, Egberto; Kaulbach, Helen; Zakaria, Marjorie; Lowell, Bradford; Radovick, Sally; Liberman, M. Charles; Wondisford, Fredric

    1999-01-01

    Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TRα and TRβ isoforms are products of 2 distinct genes, and the β1 and β2 isoforms are splice variants of the same gene. Whereas TRα1 and TRβ1 are widely expressed, expression of the TRβ2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TRβ locus (TRβ-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TRβ2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TRβ2 isoform. TRβ2-null mice have preserved expression of the TRα and TRβ1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TRβ-null mice, indicating the important role of TRβ2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TRβ2-null mice exhibit no evidence of hearing impairment, indicating that TRβ1 and TRβ2 subserve divergent roles in the regulation of auditory function. PMID:10430610

  5. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  6. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    Science.gov (United States)

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined.

  7. Two novel human NUMB isoforms provide a potential link between development and cancer.

    Science.gov (United States)

    Karaczyn, Aldona; Bani-Yaghoub, Mahmud; Tremblay, Roger; Kubu, Chris; Cowling, Rebecca; Adams, Tamara L; Prudovsky, Igor; Spicer, Douglas; Friesel, Robert; Vary, Calvin; Verdi, Joseph M

    2010-12-01

    We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  8. Two novel human NUMB isoforms provide a potential link between development and cancer

    Directory of Open Access Journals (Sweden)

    Prudovsky Igor

    2010-12-01

    Full Text Available Abstract We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif GTPase activating protein 1. Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5 and filopodia (NUMB6 in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  9. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    Science.gov (United States)

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  10. Structure- and isoform-specific glucuronidation of six curcumin analogs.

    Science.gov (United States)

    Lu, Danyi; Liu, Hui; Ye, Wencai; Wang, Ying; Wu, Baojian

    2017-04-01

    1. In the present study, we aimed to characterize the glucuronidation of six curcumin analogs (i.e. RAO-3, RAO-8, RAO-9, RAO-18, RAO-19, and RAO-23) derived from galangal using human liver microsomes (HLM) and twelve expressed UGT enzymes. 2. Formation of glucuronide was confirmed using high-resolution mass spectrometry. Single glucuronide metabolite was generated from each of six curcumin analogs. The fragmentation patterns were analyzed and were found to differ significantly between alcoholic and phenolic glucuronides. 3. All six curcumin analogs except one (RAO-23) underwent significant glucuronidation in HLM and expressed UGT enzymes. In general, the methoxy group (close to the phenolic hydroxyl group) enhanced the glucuronidation liability of the curcumin analogs. 4. UGT1A9 and UGT2B7 were primarily responsible for the glucuronidation of two alcoholic analogs (RAO-3 and RAO-18). By contrast, UGT1A9 and four UGT2Bs (UGT2B4, 2B7, 2B15 and 2B17) played important roles in conjugating three phenolic analogs (RAO-8, RAO-9, and RAO-19). Interestingly, the conjugated double bonds system (in the aliphatic chain) was crucial to the substrate selectivity of gastrointestinal UGTs (i.e. UGT1A7, 1A8 and 1A10). 5. In conclusion, glucuronidation of six curcumin analogs from galangal were structure- and isoform-specific. The knowledge should be useful in identifying a curcumin analog with improved metabolic property.

  11. Searching iron sensors in plants by exploring the link among 2’-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency

    Directory of Open Access Journals (Sweden)

    GIANPIERO eVIGANI

    2013-05-01

    Full Text Available Knowledge accumulated on the regulation of iron (Fe homeostasis, its intracellular trafficking and transport across various cellular compartments and organs in plants; storage proteins, transporters and transcription factors involved in Fe metabolism have been analysed in detail in recent years. However, the key sensor(s of cellular plant Fe status triggering the long-distance shoot-root signalling and leading to the root Fe-deficiency responses is (are still unknown. Local Fe sensing is also a major task for roots, for adjusting the internal Fe requirements to external Fe availability: how such sensing is achieved and how it leads to metabolic adjustments in case of nutrient shortage, is mostly unknown. Two proteins belonging to the 2′-OG dependent dioxygenases family accumulate several folds in Fe-deficient Arabidopsis roots. Such proteins require Fe(II as enzymatic cofactor; one of their subgroups, the HIF-P4H (Hypoxia Inducible Factor- Prolyl 4-Hydroxylase, is an effective oxygen sensor in animal cells. We envisage here the possibility that some members of the 2′-OG dioxygenase family may be involved in the Fe-deficiency response and in the metabolic adjustments to Fe deficiency or even in sensing Fe, in plant cells.

  12. Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

    Science.gov (United States)

    Nagasaki, Toshihiro; Hongo, Yuki; Koito, Tomoko; Nakamura-Kusakabe, Ikumi; Shimamura, Shigeru; Takaki, Yoshihiro; Yoshida, Takao; Maruyama, Tadashi; Inoue, Koji

    2015-03-01

    It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

  13. Spectroscopic and computational studies of NTBC bound to the non-heme iron enzyme (4-hydroxyphenyl)pyruvate dioxygenase: active site contributions to drug inhibition.

    Science.gov (United States)

    Neidig, Michael L; Decker, Andrea; Kavana, Michael; Moran, Graham R; Solomon, Edward I

    2005-12-01

    (4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an alpha-keto-acid-dependent dioxygenase which catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of tyrosine catabolism. While several di- and tri-ketone alkaloids are known as inhibitors of HPPD and used commercially as herbicides, one such inhibitor, [2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), has also been used therapeutically to treat type I tyrosinemia and alkaptonuria in humans. To gain further insight into the mechanism of inhibition by NTBC, a combination of CD/MCD spectroscopy and DFT calculations of HPPD/Fe(II)/NTBC has been performed to evaluate the contribution of the Fe(II)-NTBC bonding interaction to the high affinity of this drug for the enzyme. The results indicate that the bonding of NTBC to Fe(II) is very similar to that for HPP, both involving similar pi-backbonding interactions between NTBC/HPP and Fe(II). Combined with the result that the calculated binding energy of NTBC is, in fact, approximately 3 kcal/mol less than that for HPP, the bidentate coordination of NTBC to Fe(II) is not solely responsible for its extremely high affinity for the enzyme. Thus, the pi-stacking interactions between the aromatic rings of NTBC and two phenyalanine residues, as observed in the crystallography of the HPPD/Fe(II)/NTBC complex, appear to be responsible for the observed high affinity of drug binding.

  14. Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.

    Science.gov (United States)

    Stenflo, J; Holme, E; Lindstedt, S; Chandramouli, N; Huang, L H; Tam, J P; Merrifield, R B

    1989-01-01

    3-Hydroxyaspartic acid and 3-hydroxyasparagine are two rare amino acids that are present in domains homologous to the epidermal growth factor precursor in vitamin K-dependent plasma proteins as well as in proteins that do not require vitamin K for normal biosynthesis. They are formed by posttranslational hydroxylation of aspartic acid and asparagine, respectively. The first epidermal growth factor-like domain in factor IX (residues 45-87) was synthesized with aspartic acid in position 64, replacing 3-hydroxyaspartic acid. It was used as substrate in a hydroxylase assay with rat liver microsomes as the source of enzyme and reaction conditions that satisfy the requirements of 2-oxoglutarate-dependent dioxygenases. The synthetic peptide stimulated the 2-oxoglutarate decarboxylation in contrast to synthetic, modified epidermal growth factor (Met-21 and His-22 deleted and Glu-24 replaced by Asp) and synthetic peptides corresponding to residues 60-71 in human factor IX. This indicates that the hydroxylase is a 2-oxoglutarate-dependent dioxygenase with a selective substrate requirement. Images PMID:2492106

  15. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Chad R. [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States); Hao, Quan [MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853-8001 (United States); Stipanuk, Martha H., E-mail: mhs6@cornell.edu [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States)

    2005-11-01

    Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  16. Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

    Institute of Scientific and Technical Information of China (English)

    REN He-jun; LU Yang; ZHOU Rui; DAI Chun-yan; WANG Yan; ZHANG Lan-ying

    2012-01-01

    To find new extradiol dioxygenases(EDOs,EC 1.13.11.2),a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 ℃,and BphC_A showed more than 40% of its initial activity at 5 ℃.The activity of purified BphC_A was significantly induced by Mn2+ and slightly reduced bv Al3+,Cu2+ and Zn2+.

  17. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with novel Schiff bases: identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones.

    Science.gov (United States)

    Sarikaya, Busra; Ceruso, Mariangela; Carta, Fabrizio; Supuran, Claudiu T

    2014-11-01

    A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.

  18. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Science.gov (United States)

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  19. Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

    Directory of Open Access Journals (Sweden)

    De Santanu

    2012-01-01

    Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

  20. Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification

    Science.gov (United States)

    Shen, Jiangchuan; Keithly, Mary E.; Armstrong, Richard N.; Higgins, Khadine A.; Edmonds, Katherine A.; Giedroc, David P.

    2016-01-01

    Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus (S. aureus) and is under the transcriptional control of the persulfide sensor CstR and H2S. Here we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (Ethylmalonic Encephalopathy Protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both activities require Cys408. CstB protects S. aureus against H2S toxicity with C201S and C408S cstB genes unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts the cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon the elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S stress, in concert with the other enzymes encoded by the cst operon. PMID:26177047

  1. 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover

    Science.gov (United States)

    Kamutira, Philaiwarong; Watthaisong, Pratchaya; Thotsaporn, Kittisak; Tongsook, Chanakan; Juttulapa, Maneerat; Nijvipakul, Sarayut; Chaiyen, Pimchai

    2017-01-01

    3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k’cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology

  2. Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil.

    Science.gov (United States)

    Sawulski, Przemyslaw; Clipson, Nicholas; Doyle, Evelyn

    2014-11-01

    Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides

  3. Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities.

    Directory of Open Access Journals (Sweden)

    Luis J Cocka

    2012-09-01

    Full Text Available Tetherin (BST-2/CD317/HM1.24 is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin, which lacks 12 residues present in the long isoform (l-Tetherin, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB signaling whereas s-Tetherin does not activate NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since co-expression of s-Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into Tetherin function both in the presence and absence of infection.

  4. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

  5. Differential dynamics of RAS isoforms in GDP- and GTP-bound states.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2015-06-01

    RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange. © 2015 Wiley Periodicals, Inc.

  6. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  7. Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

    Science.gov (United States)

    Ziak, M; Meier, M; Etter, K S; Roth, J

    2001-01-12

    We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

  8. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    Directory of Open Access Journals (Sweden)

    Lennart Hilbert

    2013-10-01

    Full Text Available The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text]A, [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], [Formula: see text]-actin ([Formula: see text]A, [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], and [Formula: see text]-actin-tropomoysin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]. Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis indicated that the in vitro motility of [Formula: see text]A, [Formula: see text]A, and [Formula: see text]A-Tm[Formula: see text] is not distinguishable. Compared to these three 'baseline conditions', statistically significant differences ([Formula: see text] were: [Formula: see text]A-Tm[Formula: see text] - actin sliding velocity increased 1.12-fold, [Formula: see text]A-Tm[Formula: see text] - motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, [Formula: see text]A-Tm[Formula: see text] - run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: [Formula: see text]A-Tm[Formula: see text] - myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, [Formula: see text

  9. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

    OpenAIRE

    Erickson, J.D.; Schafer, M K; Bonner, T I; Eiden, L. E.; Weihe, E.

    1996-01-01

    A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpr...

  10. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin

    Science.gov (United States)

    Fornasa, Giulia; Tsilingiri, Katerina; Caprioli, Flavio; Botti, Fiorenzo; Mapelli, Marina; Meller, Stephan; Kislat, Andreas; Homey, Bernhard; Di Sabatino, Antonio; Sonzogni, Angelica; Viale, Giuseppe; Diaferia, Giuseppe; Gori, Alessandro; Longhi, Renato; Penna, Giuseppe; Rescigno, Maria

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. Objective In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. Methods We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. Results We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. Conclusions We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform. PMID:26014813

  11. The Effect of Exercise Training on Skeletal Muscle Glucose Transorter Isoform GLUT4 Concentration in the Obese Zucker Rat

    Science.gov (United States)

    1991-05-01

    NUMBERS The Effect of Exercise Training on Skeletal Muscle Glucose Transorter Isoform GLUT4 Concentration in the Obese Zucker Rat 6. AUTHOR(S) Eric A...Zr) THE EFFECT OF EXERCISE TRAINING ON SKELETAL MUSCLE GLUCOSE TRANSPORTER ISOFORM GLUT4 CONCENTRATION IN THE OBESE ZUCKER RAT by Eric Anthony Banks...laboratory for their help. Eric A. Banks v ABSTRACT THE EFFECT OF EXERCISE TRAINING ON SKELETAL MUSCLE GLUCOSE TRANSPORTER ISOFORM GLUT4 CONCENTRATION IN

  12. Computational Estimates of Binding Affinities for Estrogen Receptor Isoforms in Rainbow Trout

    CERN Document Server

    Shyu, Conrad

    2009-01-01

    Molecular dynamics simulations are performed to determine the binding affinities between the hormone 17 beta-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout (Oncorhynchus mykiss). Previous studies have demonstrated that a recent, unique gene duplication of the ER alpha subtype created two isoforms ER alpha 1 and ER alpha 2, and an early secondary split of ER beta produced two distinct isoforms of ER beta 1 and ER beta 2 based on the phylogenetic analysis. The objective of our computational studies is to provide insight into the underlying evolutionary selection pressure on the ER isoforms. Our results show that E2 binds preferentially to ER alpha 1. This finding corresponds to the experimental results as the ERs evolved from gene duplication events are frequently free from selective pressure and should exhibit no deleterious effects. The E2, however, only binds slightly better to ER beta 2. Both isoforms remain competitive. This finding reflects the fact that since ER beta 2 ...

  13. MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

    Science.gov (United States)

    Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

    2011-07-01

    Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

  14. Segmental distribution of myosin heavy chain isoforms within single muscle fibers.

    Science.gov (United States)

    Zhang, Ming; Gould, Maree

    2017-02-18

    Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the 3 EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I&cenveo_unknown_entity_wingdings_F0F3;IIA&cenveo_unknown_entity_wingdings_F0F3;IIX&cenveo_unknown_entity_wingdings_F0F3;IIB, however in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be due to the myonuclear domain. This article is protected by copyright. All rights reserved.

  15. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  16. The relationship between gene isoform multiplicity, number of exons and protein divergence.

    Directory of Open Access Journals (Sweden)

    Jordi Morata

    Full Text Available At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly. In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences.

  17. Quantification of various APP-mRNA isoforms and epistasis in Lesch-Nyhan disease.

    Science.gov (United States)

    Nguyen, Khue Vu; Nyhan, William L

    2017-03-16

    The present work is the development of a simple and specific kinetic method based on RT-PCR technique coupled with direct sequencing for quantification of various amyloid precursor protein-mRNA isoforms (APP-mRNA isoforms) in biological samples, especially for identifying the most abundant one that may decisive for the normal status or disease risk. Application of this kinetic method to the Lesch-Nyhan disease (LND) was performed and results indicated an epistasis between mutated hypoxanthine phosphoribosyltransferase1 (HPRT1) and APP genes. APP-mRNA isoform of 624bp, with a deletion starting after 49bp of the 5' end of exon 3 followed by a complete deletion of exons 4-15, mutations in exon 1: c.22C>T, p.L18F, and exon 3: c.269A>G, p.Q90R encoding APP207 isoform, was the most abundant one in most of the LND patients and would be responsible for the neurobehavioral syndrome in these patients. The method is useful for identifying the defective APP-mRNA isoform in LND patients, and in neurodevelopmental and neurodegenerative disorders in which the APP gene is involved in the pathogenesis of diseases such as autism, fragile X syndrome, amyotrophic lateral sclerosis, and Alzheimer's disease, and may pave the way for new strategies applicable to rational antisense drugs design. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Tau protein assembles into isoform- and disulfide-dependent polymorphic fibrils with distinct structural properties.

    Science.gov (United States)

    Furukawa, Yoshiaki; Kaneko, Kumi; Nukina, Nobuyuki

    2011-08-05

    Tauopathies are neurodegenerative diseases in which insoluble fibrillar aggregates of a microtubule-binding protein, Tau, are abnormally accumulated. Pathological Tau fibrils often exhibit structural polymorphisms that differ among phenotypically distinct tauopathies; however, a molecular mechanism to generate polymorphic Tau fibrils remains obscure. Here, we note the formation of a disulfide bond in isoforms of full-length Tau and show that the thiol-disulfide status as well as the isoform composition determines structural and morphological properties of Tau fibrils in vitro. Mainly two regions in a Tau primary sequence are found to act as structural blocks for building a protease-resistant core of Tau fibrils. Interactions among those two blocks for building a core structure depend upon the thiol-disulfide status in each isoform of Tau, which results in the formation of polymorphic fibrils with distinct structural properties. Furthermore, we have found that more diverse structures of Tau fibrils emerge through a cross-seeded fibrillation between heterologous pairs of Tau isoforms. We thus propose that isoform- and disulfide-dependent combinatorial interactions among multiple regions in a Tau sequence endow Tau fibrils with various structures, i.e. polymorphism.

  19. Tau Protein Assembles into Isoform- and Disulfide-dependent Polymorphic Fibrils with Distinct Structural Properties*

    Science.gov (United States)

    Furukawa, Yoshiaki; Kaneko, Kumi; Nukina, Nobuyuki

    2011-01-01

    Tauopathies are neurodegenerative diseases in which insoluble fibrillar aggregates of a microtubule-binding protein, Tau, are abnormally accumulated. Pathological Tau fibrils often exhibit structural polymorphisms that differ among phenotypically distinct tauopathies; however, a molecular mechanism to generate polymorphic Tau fibrils remains obscure. Here, we note the formation of a disulfide bond in isoforms of full-length Tau and show that the thiol-disulfide status as well as the isoform composition determines structural and morphological properties of Tau fibrils in vitro. Mainly two regions in a Tau primary sequence are found to act as structural blocks for building a protease-resistant core of Tau fibrils. Interactions among those two blocks for building a core structure depend upon the thiol-disulfide status in each isoform of Tau, which results in the formation of polymorphic fibrils with distinct structural properties. Furthermore, we have found that more diverse structures of Tau fibrils emerge through a cross-seeded fibrillation between heterologous pairs of Tau isoforms. We thus propose that isoform- and disulfide-dependent combinatorial interactions among multiple regions in a Tau sequence endow Tau fibrils with various structures, i.e. polymorphism. PMID:21659525

  20. Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers.

    Science.gov (United States)

    Sellin, Mikael E; Stenmark, Sonja; Gullberg, Martin

    2012-11-01

    Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform-specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks-a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.

  1. A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

    Directory of Open Access Journals (Sweden)

    Evangelos D Karousis

    Full Text Available Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02 which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

  2. Novel frataxin isoforms may contribute to the pathological mechanism of Friedreich ataxia.

    Directory of Open Access Journals (Sweden)

    Haiyan Xia

    Full Text Available Friedreich ataxia (FRDA is an inherited neurodegenerative disease caused by frataxin (FXN deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III, which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.

  3. Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

    Institute of Scientific and Technical Information of China (English)

    Wenbing WANG; Carsten M(U)LLER-TIDOW; Ping JI; Bj(o)rn STEFFEN; Ralf METZGER; Paul M. SCHNEIDER; Hartmut HALFTER; Mark SCHRADER; Wolfgang E. BERDEL; Hubert SERVE

    2005-01-01

    Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with β-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF- 1isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (n=304) and tumor-free control samples (n=56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1expression was also found in hematopoietic cells. In hematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However,acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together,these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia.

  4. Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

    Science.gov (United States)

    U, M; Miyashita, T; Shikama, Y; Tadokoro, K; Yamada, M

    2001-11-30

    Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

  5. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity.

    Science.gov (United States)

    Gardella, Kacie A; Muro, Israel; Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W

    2016-03-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies.

  6. The development of longitudinal variation of Myosin isoforms in the orbital fibers of extraocular muscles of rats.

    Science.gov (United States)

    Rubinstein, Neal A; Porter, John D; Hoh, Joseph F Y

    2004-09-01

    To examine the appearance of longitudinal variation of extraocular and embryonic myosin heavy chain (MyHC) isoforms during the development of orbital singly innervated fibers of rat extraocular muscles (EOMs). EOMs were dissected from rat pups of various ages and stained with isoform-specific monoclonal antibodies to the embryonic and extraocular MyHC isoforms and to neurofilaments, as well as with labeled alpha-bungarotoxin. The orbital layers of whole muscles were examined by confocal microscopy. RNase protection assays for the embryonic (Myh3) and extraocular (Myh13) MyHC isoform mRNAs were also performed. At 10 days postpartum, the EOM MyHC RNA was first detected by RNase protection assay. At 11 days postpartum, the extraocular isoform was detected in the orbital fibers as two thin stripes just proximal and distal to the neuromuscular junction (NMJ). Over the next few weeks, the area occupied by the extraocular isoform increased to include the entire central region of the orbital fibers at and surrounding the NMJ. At the same time, the embryonic isoform became excluded from the region of the NMJ. The orbital layer fibers of rat EOMs contain a longitudinal variation in MyHC isoforms not seen in other skeletal muscles. Development of this longitudinal variation begins as a late event postpartum; and the first appearance of it may be closely linked to neural contact. This targeting of MyHC isoforms to distinct domains is unique to EOMs. Copyright Association for Research in Vision and Ophthalmology

  7. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

    Directory of Open Access Journals (Sweden)

    Jean-Simon eFortin

    2013-12-01

    Full Text Available The peptide repertoire (peptidome associated with MHC class II molecules (MHCIIs is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  8. The respiratory chain supercomplex organization is independent of COX7a2l isoforms.

    Science.gov (United States)

    Mourier, Arnaud; Matic, Stanka; Ruzzenente, Benedetta; Larsson, Nils-Göran; Milenkovic, Dusanka

    2014-12-01

    The organization of individual respiratory chain complexes into supercomplexes or respirasomes has attracted great interest because of the implications for cellular energy conversion. Recently, it was reported that commonly used mouse strains harbor a short COX7a2l (SCAFI) gene isoform that supposedly precludes the formation of complex IV-containing supercomplexes. This claim potentially has serious implications for numerous mouse studies addressing important topics in metabolism, including adaptation to space flights. Using several complementary experimental approaches, we show that mice with the short COX7a2l isoform have normal biogenesis and steady-state levels of complex IV-containing supercomplexes and consequently have normal respiratory chain function. Furthermore, we use a mouse knockout of Lrpprc and show that loss of complex IV compromises respirasome formation. We conclude that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not prevent their use in metabolism research.

  9. Epidermal growth-factor-induced transcript isoform variation drives mammary cell migration.

    Directory of Open Access Journals (Sweden)

    Wolfgang J Köstler

    Full Text Available Signal-induced transcript isoform variation (TIV includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF. We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.

  10. Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes

    DEFF Research Database (Denmark)

    Kamstrup, Pia Rørbæk; Nordestgaard, Børge

    2013-01-01

    BACKGROUND: Low concentrations of lipoprotein(a) in plasma are associated with increased risk of type 2 diabetes, but whether this association is causal is unclear. Variations in the LPA gene affect lipoprotein(a) isoform size and concentrations in plasma. We therefore did a Mendelian randomisation...... study to investigate whether large isoform size, low concentrations in plasma, or both, are causally associated with type 2 diabetes. METHODS: We assessed data for adults from the Danish general population enrolled in the Copenhagen City Heart Study and the Copenhagen General Population Study......, with and without type 2 diabetes. Eligible participants had data for lipoprotein(a) concentrations in plasma, LPA kringle IV type 2 (KIV-2) sums of repeats (affecting both isoform size and plasma concentrations), and carrier status for the LPA single-nucleotide polymorphism rs10455872 (mainly affecting...

  11. Exposing the Specific Roles of the Invariant Chain Isoforms in Shaping the MHC Class II Peptidome.

    Science.gov (United States)

    Fortin, Jean-Simon; Cloutier, Maryse; Thibodeau, Jacques

    2013-12-13

    The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  12. The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

    Science.gov (United States)

    Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

    2017-06-26

    Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

  13. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes...... a non-functional FR beta on the surface, and, in addition, nanomolar concentrations of a secretory functional FBP (29 kDa) can be present in the secondary granules. A statistically significant correlation between the concentrations of functional FBP, probably a gamma isoform, in granulocytes and serum......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...

  14. New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents

    Directory of Open Access Journals (Sweden)

    Hanna Szaefer

    2013-08-01

    Full Text Available Cytochromes P450 (P450 are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms’ genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1 that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

  15. The Invasion and Metastasis Promotion Role of CD97 Small Isoform in Gastric Carcinoma

    DEFF Research Database (Denmark)

    Liu, Daren; Trojanowicz, Bogusz; Ye, Longyun

    2012-01-01

    CD97 is over-expressed in the majority of gastric adenocarcinomas and is associated with its dedifferentiation and aggressiveness. Our previous results demonstrated that out of three CD97 isoforms tested, only the small one was able to promote increased invasiveness in vitro. Based on these data we...... further aimed to investigate the role of CD97 small isoform in gastric cancer progression in vivo by employing the cells with a stable CD97 small isoform knock-down and an orthotopic gastric cancer mouse model. We could demonstrate that the knock down of CD97/EGF1,2,5, led to a significant decrease...... in the number of cells penetrating the gelatin coated membrane as compared with control cells. In the gastric cancer mouse model, both the hypodermic and the orthotopic yielded tumor masses of the CD97/EGF1,2,5kd group and were significantly smaller than the control. Metastatic tumor cell number in early...

  16. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    Science.gov (United States)

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.

  17. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

    Science.gov (United States)

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J; Vacic, Vladimir; Calderwood, Michael A; Roth, Frederick P; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M

    2014-04-11

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

  18. Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites in Infectious Diseases: Potential Role in Macrophage-Mediated Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Yuki Murakami

    2013-01-01

    Full Text Available Indoleamine 2,3-dioxygenase 1 (IDO1, the L-tryptophan-degrading enzyme, plays a key role in the immunomodulatory effects on several types of immune cells. Originally known for its regulatory function during pregnancy and chronic inflammation in tumorigenesis, the activity of IDO1 seems to modify the inflammatory state of infectious diseases. The pathophysiologic activity of L-tryptophan metabolites, kynurenines, is well recognized. Therefore, an understanding of the regulation of IDO1 and the subsequent biochemical reactions is essential for the design of therapeutic strategies in certain immune diseases. In this paper, current knowledge about the role of IDO1 and its metabolites during various infectious diseases is presented. Particularly, the regulation of type I interferons (IFNs production via IDO1 in virus infection is discussed. This paper offers insights into new therapeutic strategies in the modulation of viral infection and several immune-related disorders.

  19. Safety, immune and clinical responses in metastatic melanoma patients vaccinated with a long peptide derived from indoleamine 2,3-dioxygenase in combination with ipilimumab

    DEFF Research Database (Denmark)

    Bjoern, Jon; Iversen, Trine Zeeberg; Nitschke, Nikolaj Juul;

    2016-01-01

    BACKGROUND AIM: Indoleamine 2,3-dioxygenase (IDO) is an emerging new target in cancer therapy that can be targeted with active immunotherapy (e.g. through peptide vaccination). Furthermore, IDO has been identified as a key mechanism underlying resistance to treatment with the checkpoint blocking...... antibody ipilimumab (ipi). METHODS: Ten patients with metastatic melanoma participated in a phase I first-in-human clinical study assessing safety of combining ipi with a 21-mer synthetic peptide vaccine from IDO denoted IDOlong. Secondary and tertiary end points included vaccine and clinical response....... RESULTS: Treatment was generally safe and well tolerated. Vaccine related adverse reactions included grade I and II erythema, oedema and pruritus at the vaccination site, which were manageable with mild topical corticosteroids. One patient developed presumed ipi-induced colitis. It initially responded...

  20. Inhibition of indoleamine 2,3-dioxygenase 1/2 prevented cognitive impairment and energetic metabolism changes in the hippocampus of adult rats subjected to polymicrobial sepsis.

    Science.gov (United States)

    Comim, Clarissa M; Freiberger, Viviane; Ventura, Letícia; Mina, Francielle; Ferreira, Gabriela K; Michels, Monique; Generoso, Jaqueline S; Streck, Emílio L; Quevedo, João; Barichello, Tatiana; Dal-Pizzol, Felipe

    2017-04-15

    Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection that may affect the brain. We investigated the role of indoleamine 2,3-dioxygenase (IDO-1/2) inhibition on long-term memory and energetic metabolism after experimental sepsis by caecal ligation and perforation (CLP). Experimental sepsis increased the activity of complexes I, II-III and IV at 24h after CLP, and IDO-1/2 inhibition normalized the activity of these complexes in the hippocampus. Wistar rats presented impairment of habituation and aversive memories 10days after CLP. Adjuvant treatment with the IDO inhibitor prevented long-term cognitive impairment triggered by sepsis. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Interferon-γ regulates the proliferation and differentiation of mesenchymal stem cells via activation of indoleamine 2,3 dioxygenase (IDO.

    Directory of Open Access Journals (Sweden)

    Juliana Croitoru-Lamoury

    Full Text Available The kynurenine pathway (KP of tryptophan metabolism is linked to antimicrobial activity and modulation of immune responses but its role in stem cell biology is unknown. We show that human and mouse mesenchymal and neural stem cells (MSCs and NSCs express the complete KP, including indoleamine 2,3 dioxygenase 1 (IDO and IDO2, that it is highly regulated by type I (IFN-β and II interferons (IFN-γ, and that its transcriptional modulation depends on the type of interferon, cell type and species. IFN-γ inhibited proliferation and altered human and mouse MSC neural, adipocytic and osteocytic differentiation via the activation of IDO. A functional KP present in MSCs, NSCs and perhaps other stem cell types offers novel therapeutic opportunities for optimisation of stem cell proliferation and differentiation.

  2. In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

    Directory of Open Access Journals (Sweden)

    Kristin R Wildsmith

    Full Text Available Apolipoprotein E (ApoE is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4 each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD. Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS, we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

  3. Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

    Science.gov (United States)

    Pires, Susana S; Hughes, Steven; Turton, Michael; Melyan, Zare; Peirson, Stuart N; Zheng, Lei; Kosmaoglou, Maria; Bellingham, James; Cheetham, Michael E; Lucas, Robert J; Foster, Russell G; Hankins, Mark W; Halford, Stephanie

    2009-09-30

    Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

  4. IQSeq: integrated isoform quantification analysis based on next-generation sequencing.

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    Jiang Du

    Full Text Available With the recent advances in high-throughput RNA sequencing (RNA-Seq, biologists are able to measure transcription with unprecedented precision. One problem that can now be tackled is that of isoform quantification: here one tries to reconstruct the abundances of isoforms of a gene. We have developed a statistical solution for this problem, based on analyzing a set of RNA-Seq reads, and a practical implementation, available from archive.gersteinlab.org/proj/rnaseq/IQSeq, in a tool we call IQSeq (Isoform Quantification in next-generation Sequencing. Here, we present theoretical results which IQSeq is based on, and then use both simulated and real datasets to illustrate various applications of the tool. In order to measure the accuracy of an isoform-quantification result, one would try to estimate the average variance of the estimated isoform abundances for each gene (based on resampling the RNA-seq reads, and IQSeq has a particularly fast algorithm (based on the Fisher Information Matrix for calculating this, achieving a speedup of ~ 500 times compared to brute-force resampling. IQSeq also calculates an information theoretic measure of overall transcriptome complexity to describe isoform abundance for a whole experiment. IQSeq has many features that are particularly useful in RNA-Seq experimental design, allowing one to optimally model the integration of different sequencing technologies in a cost-effective way. In particular, the IQSeq formalism integrates the analysis of different sample (i.e. read sets generated from different technologies within the same statistical framework. It also supports a generalized statistical partial-sample-generation function to model the sequencing process. This allows one to have a modular, "plugin-able" read-generation function to support the particularities of the many evolving sequencing technologies.

  5. Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein

    Science.gov (United States)

    Minervini, Giovanni; Mazzotta, Gabriella M.; Masiero, Alessandro; Sartori, Elena; Corrà, Samantha; Potenza, Emilio; Costa, Rodolfo; Tosatto, Silvio C. E.

    2015-01-01

    Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization. PMID:26211615

  6. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  7. Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure.

    Science.gov (United States)

    Bousfield, George R; Butnev, Vladimir Y; Bidart, Jean-Michel; Dalpathado, Dilusha; Irungu, Janet; Desaire, Heather

    2008-02-12

    Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the

  8. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

    Science.gov (United States)

    Bridges, Hannah R; Mohammed, Khairunnisa; Harbour, Michael E; Hirst, Judy

    2017-03-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

  9. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    Science.gov (United States)

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

  10. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  11. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    Directory of Open Access Journals (Sweden)

    Tanja Seeger

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521 showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

  12. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    Science.gov (United States)

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  13. The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

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    Lili Li

    Full Text Available Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB(αΔ mice. Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ mice. CREB(αΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ. CREB(αΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

  14. Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana.

    Science.gov (United States)

    Kai, Kosuke; Mizutani, Masaharu; Kawamura, Naohiro; Yamamoto, Ryotaro; Tamai, Michiko; Yamaguchi, Hikaru; Sakata, Kanzo; Shimizu, Bun-ichi

    2008-09-01

    Coumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its beta-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3'-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2OGD) family was designated as F6'H1. T-DNA insertion mutants of F6'H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6'H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6'H1 protein exhibited ortho-hydroxylase activity for feruloyl CoA (K(m) = 36.0 +/- 4.27 microM; k(cat) = 11.0 +/- 0.45 sec(-1)) to form 6'-hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)- and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.

  15. Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

    Science.gov (United States)

    Dionisi, Hebe M.; Chewning, Christopher S.; Morgan, Katherine H.; Menn, Fu-Min; Easter, James P.; Sayler, Gary S.

    2004-01-01

    We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene. PMID:15240274

  16. Comparative quantitative prevalence of mycobacteria and functionally abundant nidA, nahAc, and nagAc Dioxygenase genes in coal tar contaminated sediments

    Energy Technology Data Exchange (ETDEWEB)

    Jennifer M. DeBruyn; Christopher S. Chewning; Gary S. Sayler [University of Tennessee, Knoxville, TN (United States). Department of Ecology and Evolutionary Biology

    2007-08-01

    The Chattanooga Creek Superfund site is heavily contaminated with metals, pesticides, and coal tar with sediments exhibiting high concentrations of polycyclic aromatic hydrocarbons (PAHs). High molecular weight PAHs are of concern because of their toxicity and recalcitrance in the environment; as such, there is great interest in microbes, such as fast-growing Mycobacterium spp., capable of degradation of these compounds. Real-time quantitative PCR assays were developed targeting multiple dioxygenase genes to assess the ecology and functional diversity of PAH-degrading communities. These assays target the Mycobacterium nidA, {beta}-proteobacteria nagAc, and {gamma}-proteobacteria nahAc with the specific goal of testing the hypothesis that Mycobacteria catabolic genes are enriched and may be functionally associated with high molecular weight PAH biodegradation in Chattanooga Creek. Dioxygenase gene abundances were quantitatively compared to naphthalene and pyrene mineralization, and temporal and spatial PAH concentrations. nidA abundances ranged from 5.69 x 10{sup 4} to 4.92 x 10{sup 6} copies per gram sediment; nagAc from 2.42 x 10{sup 3} to 1.21 x 10{sup 7}, and nahAc from below detection to 4.01 x 10{sup 6} copies per gram sediment. There was a significantly greater abundance of nidA and nagAc at sites with the greatest concentrations of PAHs. In addition, nidA and nagAc were significantly positively correlated, indicating a coexistence of organisms carrying these genes. A positive relationship was also observed between nidA and nagAc and pyrene mineralization indicating that these genes serve as biomarkers for pyrene degradation. A 16S rDNA clone library of fast-growing Mycobacteria indicated that the population is very diverse and likely plays an important role in attenuation of high molecular weight PAHs from Chattanooga Creek. 35 refs., 5 figs., 1 tab.

  17. Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

    Science.gov (United States)

    Singh, Vipender; Fedeles, Bogdan I; Li, Deyu; Delaney, James C; Kozekov, Ivan D; Kozekova, Albena; Marnett, Lawrence J; Rizzo, Carmelo J; Essigmann, John M

    2014-09-15

    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

  18. Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS 3.

    Science.gov (United States)

    Schweizer, D; Markus, A; Seez, M; Ruf, H H; Lingens, F

    1987-07-05

    4-Chlorophenylacetate 3,4-dioxygenase system from Pseudomonas sp. CBS 3 consists of two protein components, a red-brown iron-sulfur protein (component A) which functions as dioxygenase and an orange-colored reductase (component B). Component B was purified by a five-step procedure. Criterion of purity was sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which also showed that the protein consists of one polypeptide species with a molecular weight of 35,000. With gel chromatography on Sephadex G-100 also, a molecular weight of 35,000 was found for the native enzyme, suggesting a monomeric structure for the reductase enzyme. The isoelectric point was determined as pH 4.8. The visible absorption spectrum of the homogeneous protein exhibits maxima at 336, 394, and 458 nm. One mol of FMN, 2.1 mol of iron, and 1.7 mol of acid-labile sulfide were found in one mol of component B. The EPR-spectrum of the reduced form of the enzyme (with NADH) showed two types of signals. The signal at g values of 2.03, 1.94, and 1.90 was assigned to an iron-sulfur cluster of the [2Fe-2S]-type. The properties of the other signal type at g = 2.004 are characteristic of flavosemiquinone radical which does not show coupling to an other paramagnetic center. The apparent Km values for 2,6-dichlorophenolindophenol, cytochrome c, and NADH were calculated from Lineweaver-Burk plots as 6.3, 2.3, and 32 microM, respectively. Inhibitors of the reductase are metal-chelating reagents and sulfhydryl-inhibiting compounds. Component B reduces several redox compounds: 2,6-dichlorophenolindophenol, potassium hexacyanoferrat III, cytochrome c, methylene blue, and nitro blue tetrazolium.

  19. Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain.

    Directory of Open Access Journals (Sweden)

    Wei Wu

    Full Text Available To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD, the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO, and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

  20. [Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Condtions].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Lyashchenko, M S; Gataullinaa, M O; Kompantseva, E I

    2016-01-01

    Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

  1. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  2. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

    Directory of Open Access Journals (Sweden)

    Pio Ruben

    2010-12-01

    Full Text Available Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189 have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p xxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033 between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken

  3. Changes in claudin isoform expression in the gill during salinity shifts and smoltification of Atlantic salmon

    DEFF Research Database (Denmark)

    Madsen, Steffen; Tipsmark, Christian Kølbæk

    2008-01-01

    Permeability changes of the gill epithelium of euryhaline teleosts are imperative during salinity acclimation. This study examined the expression patterns of 5 claudin isoforms in the salmon gill. These are membrane proteins being major determinants of selective and gross tight junction permeabil......Permeability changes of the gill epithelium of euryhaline teleosts are imperative during salinity acclimation. This study examined the expression patterns of 5 claudin isoforms in the salmon gill. These are membrane proteins being major determinants of selective and gross tight junction...

  4. Differential ligand binding affinities of human estrogen receptor-α isoforms

    OpenAIRE

    Amanda H.Y. Lin; Li, Rachel W. S.; Ho, Eva Y. W.; George P H Leung; Susan W S Leung; Paul M Vanhoutte; Man, Ricky Y K

    2013-01-01

    Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cel...

  5. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    Directory of Open Access Journals (Sweden)

    Thomas Geoffrey C

    2011-05-01

    Full Text Available Abstract Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA comprises ~530 residues, the G isoform (MSG is ~730 residues, and this third isoform (MSH-halophilic is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C

  6. Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease.

    Science.gov (United States)

    Boczonadi, Veronika; Giunta, Michele; Lane, Maria; Tulinius, Mar; Schara, Ulrike; Horvath, Rita

    2015-06-01

    Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues. We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  7. Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

    Science.gov (United States)

    Andruchova, Olena; Stephenson, Gabriela M M; Andruchov, Oleg; Stephenson, D George; Galler, Stefan

    2006-07-01

    Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

  8. Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen.

    Science.gov (United States)

    Schenk, Martijn F; Cordewener, Jan H G; America, Antoine H P; Van't Westende, Wendy P C; Smulders, Marinus J M; Gilissen, Luud J W J

    2009-03-03

    Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives. Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

  9. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    Science.gov (United States)

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  10. CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients

    NARCIS (Netherlands)

    Birzele, F.; Voss, E.; Nopora, A.; Honold, K.; Heil, F.; Lohmann, S.; Verheul, H.; Tourneau, C. Le; Delord, J.P.; Herpen, C.M. van; Mahalingam, D.; Coveler, A.L.; Meresse, V.; Weigand, S.; Runza, V.; Cannarile, M.

    2015-01-01

    PURPOSE: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform

  11. RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFAxxxb isoforms.

    Science.gov (United States)

    Bridgett, Stephen; Dellett, Margaret; Simpson, David A

    2017-12-01

    Vascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA121, VEGFA165 and VEGFA189. A second set of anti-angiogenic isoforms termed VEGFAxxxb that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFAxxxb isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFAxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7-8 junction in 10 tissues did not identify any VEGFAxxxb transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFAxxxb isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA145 and VEGFA148 isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.

  12. Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

    NARCIS (Netherlands)

    Ozgen, Hande; Kahya, Nicoletta; de Jonge, Jenny C.; Smith, Graham S. T.; Harauz, George; Hoekstra, Dick; Baron, Wia

    2014-01-01

    The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are target

  13. Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

    Science.gov (United States)

    The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) [Lp(a)], Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examinati...

  14. The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

    Science.gov (United States)

    Cao, Yanyun; Zhao, Luping; Ying, Yusang; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-06-15

    Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH.

  15. The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

    Science.gov (United States)

    Lambrechts, A; Verschelde, J L; Jonckheere, V; Goethals, M; Vandekerckhove, J; Ampe, C

    1997-02-03

    We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

  16. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

    2013-01-01

    positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O...

  17. Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis.

    Science.gov (United States)

    Yuan, Xianghe; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

    2016-02-06

    The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50-60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0-8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat) were determined by using a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.

  18. Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2

    DEFF Research Database (Denmark)

    Lee, Yun-Han; Seo, Daekwan; Choi, Kyung-Ju

    2014-01-01

    Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit H...

  19. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

    NARCIS (Netherlands)

    Rennel, E.; Waine, E.; Guan, H.; Schuler, Y.; Leenders, W.P.J.; Woolard, J.; Sugiono, M.; Gillatt, D.; Kleinerman, E.; Bates, D.; Harper, S.

    2008-01-01

    Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the

  20. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity.

    Science.gov (United States)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T; Clausen, Henrik; Linstedt, Adam D

    2014-10-31

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

  1. γIsoform-Selective Changes in PKC Immunoreactivity after Trace Eyeblink Conditioning in the Rabbit Hippocampus

    NARCIS (Netherlands)

    Zee, E.A. van der; Kronforst-Collins, M.A.; Maizels, E.T.; Hunzicker-Dunn, M.; Disterhoft, J.F.

    1997-01-01

    An immunocytochemical examination of the rabbit hippocampus was done to determine which of the Ca2+-dependent protein kinase C (PKC) isoforms (PKCα, -βI, -βII, or -γ) are involved in associative learning. The hippocampally dependent trace eyeblink conditioning task was used for behavioral training,

  2. Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-beta Isoforms for Early and Differential Dementia Diagnosis

    NARCIS (Netherlands)

    Struyfs, Hanne; Van Broeck, Bianca; Timmers, Maarten; Fransen, Erik; Sleegers, Kristel; Van Broeckhoven, Christine; De Deyn, Peter P.; Streffer, Johannes R.; Mercken, Marc; Engelborghs, Sebastiaan

    2015-01-01

    Background: Overlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimer's disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-beta (A beta) isoforms might improve the AD versus non-AD differential diagnosis. Objective: To de

  3. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    Science.gov (United States)

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

  4. Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.

    NARCIS (Netherlands)

    Sahlmuller, M.C.; Strating, J.R.P.M.; Beck, R.; Eckert, P.; Popoff, V.; Haag, M.; Hellwig, A.; Berger, I.; Brugger, B.; Wieland, F.T.

    2011-01-01

    COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in

  5. Role of PRMTs in cancer: Could minor isoforms be leaving a mark?

    Institute of Scientific and Technical Information of China (English)

    R; Mitchell; Baldwin; Alan; Morettin; Jocelyn; C?té

    2014-01-01

    Protein arginine methyltransferases(PRMTs) catalyze the methylation of a variety of protein substrates, many of which have been linked to the development, progression and aggressiveness of different types of cancer. Moreover, aberrant expression of PRMTs has been observed in several cancer types. While the link between PRMTs and cancer is a relatively new area of interest, the functional implications documented thus far warrant further investigations into its therapeutic potential. However, the expression of these enzymes and the regulation of their activity in cancer are still significantly understudied. Currently there are nine main members of the PRMT family. Further, the existence of alternatively spliced isoforms for several of these family members provides an additional layer of complexity. Specifically, PRMT1, PRMT2, CARM1 and PRMT7 have been shown to have alternative isoforms and others may be currently unrealized. Our knowledge with respect to the relative expression and the specific functions of these isoforms is largely lacking and needs attention. Here we present a review of the current knowledge of theknown alternative PRMT isoforms and provide a rationale for how they may impact on cancer and represent potentially useful targets for the development of novel therapeutic strategies.

  6. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Directory of Open Access Journals (Sweden)

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intrac