Science.gov

Sample records for promiscuous sulfatase reaction

  1. Promiscuous sulfatase activity and thio-effects in a phosphodiesterase of the alkaline phosphatase superfamily†

    PubMed Central

    Lassila, Jonathan K.; Herschlag, Daniel

    2009-01-01

    The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn2+ bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with kcat/KM value of 2 × 10−5 M−1s−1, similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these non-cognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn2+ ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 102 of the 107-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn2+ ions may play an important role in defining the selectivity of the enzymes. PMID:18975918

  2. Recent developments in enzyme promiscuity for carbon-carbon bond-forming reactions.

    PubMed

    Miao, Yufeng; Rahimi, Mehran; Geertsema, Edzard M; Poelarends, Gerrit J

    2015-04-01

    Numerous enzymes have been found to catalyze additional and completely different types of reactions relative to the natural activity they evolved for. This phenomenon, called catalytic promiscuity, has proven to be a fruitful guide for the development of novel biocatalysts for organic synthesis purposes. As such, enzymes have been identified with promiscuous catalytic activity for, one or more, eminent types of carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations. This review focuses on enzymes that promiscuously catalyze these reaction types and exhibit high enantioselectivities (in case chiral products are obtained). PMID:25598537

  3. Promiscuous words

    PubMed Central

    2013-01-01

    Promiscuity is frequently used to describe animal mating behaviour, and especially to describe multiple mating by females. Yet this use of the term is incorrect, perhaps reflecting an erroneous adoption of common language to pique reader interest. We evaluated the patterns of use and misuse of the word ‘promiscuity’ in a representative journal of animal behaviour. This survey highlights how inappropriately the term is used, and how it can conceal critical features of animal mating strategies with intriguing evolutionary significance. Further analysis of the scientific impact of papers identified by the term promiscuous or polyandrous revealed that the former were cited less frequently. We argue that using promiscuity to describe animal mating strategies is anthropomorphic, inaccurate, and potentially misleading. Consistent with other biological disciplines, the word promiscuity should be used to describe indiscriminate mating behaviour only, and that polygyny and polyandry should be used to describe male and female mating frequency respectively. PMID:24209457

  4. Triesterase and promiscuous diesterase activities of a di-Co(II)-containing organophosphate degrading enzyme reaction mechanisms.

    PubMed

    Alberto, Marta E; Pinto, Gaspar; Russo, Nino; Toscano, Marirosa

    2015-02-23

    The reaction mechanism for the hydrolysis of trimethyl phosphate and of the obtained phosphodiester by the di-Co(II) derivative of organophosphate degrading enzyme from Agrobacterium radiobacter P230(OpdA), have been investigated at density functional level of theory in the framework of the cluster model approach. Both mechanisms proceed by a multistep sequence and each catalytic cycle begins with the nucleophilic attack by a metal-bound hydroxide on the phosphorus atom of the substrate, leading to the cleavage of the phosphate-ester bond. Four exchange-correlation functionals were used to derive the potential energy profiles in protein environments. Although the enzyme is confirmed to work better as triesterase, as revealed by the barrier heights in the rate-limiting steps of the catalytic processes, its promiscuous ability to hydrolyze also the product of the reaction has been confirmed. The important role played by water molecules and some residues in the outer coordination sphere has been elucidated, while the binuclear Co(II) center accomplishes both structural and catalytic functions. To correctly describe the electronic configuration of the d shell of the metal ions, high- and low-spin arrangement jointly with the occurrence of antiferromagnetic coupling, have been herein considered. PMID:25582757

  5. Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

    PubMed Central

    2016-01-01

    The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

  6. Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

    PubMed

    Pabis, Anna; Duarte, Fernanda; Kamerlin, Shina C L

    2016-06-01

    The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

  7. The Important Roles of Steroid Sulfatase and Sulfotransferases in Gynecological Diseases

    PubMed Central

    Rižner, Tea Lanišnik

    2016-01-01

    Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by 17-ketosteroid reductases, while estradiol can also be formed from dehydroepiandrosterone by the sequential actions of 3β-hydroxysteroid dehydrogenase-Δ4-isomerase, aromatase, and 17-ketosteroid reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis. PMID:26924986

  8. The Important Roles of Steroid Sulfatase and Sulfotransferases in Gynecological Diseases.

    PubMed

    Rižner, Tea Lanišnik

    2016-01-01

    Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by 17-ketosteroid reductases, while estradiol can also be formed from dehydroepiandrosterone by the sequential actions of 3β-hydroxysteroid dehydrogenase-Δ(4)-isomerase, aromatase, and 17-ketosteroid reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis. PMID:26924986

  9. SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

    PubMed Central

    Fraldi, Alessandro; Biffi, Alessandra; Lombardi, Alessia; Visigalli, Ilaria; Pepe, Stefano; Settembre, Carmine; Nusco, Edoardo; Auricchio, Alberto; Naldini, Luigi; Ballabio, Andrea; Cosma, Maria Pia

    2007-01-01

    Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies. PMID:17206939

  10. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily.

    PubMed

    Baas, Bert-Jan; Zandvoort, Ellen; Geertsema, Edzard M; Poelarends, Gerrit J

    2013-05-27

    Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed. PMID:23649962

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

    PubMed

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

    2015-03-23

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which has a catalytic N-terminal proline residue (Pro1), can promiscuously catalyze various carbon-carbon bond-forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael-type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ-nitroaldehydes. To gain insight into how 4-OT catalyzes these unnatural reactions, we carried out exchange studies in D2 O, and X-ray crystallography studies. The former established that H-D exchange within acetaldehyde is catalyzed by 4-OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4-OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4-OT-catalyzed aldol and Michael-type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1-dependent formation of an enamine intermediate. PMID:25728471

  12. Genetics Home Reference: multiple sulfatase deficiency

    MedlinePlus

    ... condition that mainly affects the brain, skin, and skeleton. Because the signs and symptoms of multiple sulfatase ... of cells in particular tissues, specifically the brain, skeleton, and skin, cause many of the signs and ...

  13. Arylsulfatase K, a Novel Lysosomal Sulfatase*

    PubMed Central

    Wiegmann, Elena Marie; Westendorf, Eva; Kalus, Ina; Pringle, Thomas H.; Lübke, Torben; Dierks, Thomas

    2013-01-01

    The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18–22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (∼4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases. PMID:23986440

  14. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination. PMID:26244568

  15. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGESBeta

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  16. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    SciTech Connect

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  17. Mechanistic Diversity in the RuBisCO Superfamily: RuBisCO from Rhodospirillum rubrum is Not Promiscuous for Reactions Catalyzed by RuBisCO-Like Proteins (RLPs)†

    PubMed Central

    Warlick, Benjamin P. E.; Imker, Heidi J.; Sriram, Jaya; Tabita, F. Robert; Gerlt, John A.

    2012-01-01

    D-Ribulose 1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) are promiscuous, catalyzing not only carboxylation and oxygenation of D-ribulose 1,5-bisphosphate but also other promiscuous, presumably nonphysiological, reactions initiated by abstraction of the 3-proton of D-ribulose 1,5-bisphosphate. Also, RuBisCO has homologues that do not catalyze carboxylation; these are designated RuBisCO-like proteins or RLPs. Members of the two families of RLPs catalyze reactions in the recycling of 5′-methylthioadenosine (MTA) generated by polyamine synthesis: 1) the 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) “enolase” reaction in the well-known “methionine salvage” pathway in species of Bacilli; and 2) the 5-methylthio-D-ribulose 1-phosphate (MTRu 1-P) 1,3-isomerase reaction in the recently discovered “MTA-isoprenoid shunt” that generates 1-deoxy-D-xylulose 5-phosphate (DXP) for nonmevalonate isoprene synthesis in Rhodospirillum rubrum. We first studied the structure and reactivity of DK-MTP 1-P which was reported to decompose rapidly [Ashida, H., Saito, Y., Kojima, C., and Yokota, A. (2008) Biosci Biotechnol Biochem 72, 959–67]. The 2-carbonyl group of DK-MTP 1-P is rapidly hydrated in solution and can undergo enolization both nonenzymatically and enzymatically via the small amount of unhydrated material that is present. We then examined the ability of RuBisCO from R. rubrum to catalyze both of the RLP-catalyzed reactions. Contrary to a previous report [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) Science 302, 286–290], we were unable to confirm that this RuBisCO catalyzes the DK-MTP 1-P “enolase” reaction either in vitro or in vivo. We also determined that this RuBisCO does not catalyze the MTRu 1-P 1,3-isomerase reaction in vitro. Thus, although RuBisCOs can be functionally promiscuous, RuBisCO from R. rubrum is not promiscuous for either of the known RLP-catalyzed reactions. PMID:23110715

  18. Multiple sulfatase deficiency with neonatal manifestation.

    PubMed

    Garavelli, Livia; Santoro, Lucia; Iori, Alexandra; Gargano, Giancarlo; Braibanti, Silvia; Pedori, Simona; Melli, Nives; Frattini, Daniele; Zampini, Lucia; Galeazzi, Tiziana; Padella, Lucia; Pepe, Stefano; Wischmeijer, Anita; Rosato, Simonetta; Ivanovski, Ivan; Iughetti, Lorenzo; Gelmini, Chiara; Bernasconi, Sergio; Superti-Furga, Andrea; Ballabio, Andrea; Gabrielli, Orazio

    2014-01-01

    Multiple Sulfatase Deficiency (MSD; OMIM 272200) is a rare autosomal recessive inborn error of metabolism caused by mutations in the sulfatase modifying factor 1 gene, encoding the formylglycine-generating enzyme (FGE), and resulting in tissue accumulation of sulfatides, sulphated glycosaminoglycans, sphingolipids and steroid sulfates. Less than 50 cases have been published so far. We report a new case of MSD presenting in the newborn period with hypotonia, apnoea, cyanosis and rolling eyes, hepato-splenomegaly and deafness. This patient was compound heterozygous for two so far undescribed SUMF1 mutations (c.191C > A; p.S64X and c.818A > G; p.D273G). PMID:25516103

  19. Multiple sulfatase deficiency: A case series of four children

    PubMed Central

    Incecik, Faruk; Ozbek, Mehmet N.; Gungor, Serdal; Pepe, Stefano; Herguner, Ozlem M.; Mungan, Neslihan Onenli; Gungor, Sabiha; Altunbasak, Sakir

    2013-01-01

    Multiple sulfatase deficiency is biochemically characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. The gene sulfatase-modifying factor 1 (SUMF1), recently identified, encodes the enzyme responsible for post-translational modification of a cysteine residue, which is essential for the activity of sulfatases. We describe clinical findings and mutation analysis of four patients. The patients presented with hypotonia, developmental delay, coarse face, ichthyosis, and hepatosplenomegaly. The diagnosis was made through clinical findings, enzymatic assays, and mutation analysis. We were detected to be homozygous for a novel missense mutation c. 739G > C causing a p.G247R amino acid substitution in the SUMF1 protein. PMID:24339620

  20. Phylogeny of Algal Sequences Encoding Carbohydrate Sulfotransferases, Formylglycine-Dependent Sulfatases, and Putative Sulfatase Modifying Factors

    PubMed Central

    Ho, Chai-Ling

    2015-01-01

    Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs) while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs). Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs) is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs, and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute toward future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering. PMID:26635861

  1. Steroid promiscuity: Diversity of enzyme action. Preface.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri; Mason, J Ian

    2015-07-01

    This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity, dwells on the growing realization that the 'one ligand, one binding site' and 'one enzyme, one reaction' concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled 'Steroid/Sterol Signaling' . PMID:25596328

  2. Why Polyphenols have Promiscuous Actions? An Investigation by Chemical Bioinformatics.

    PubMed

    Tang, Guang-Yan

    2016-05-01

    Despite their diverse pharmacological effects, polyphenols are poor for use as drugs, which have been traditionally ascribed to their low bioavailability. However, Baell and co-workers recently proposed that the redox potential of polyphenols also plays an important role in this, because redox reactions bring promiscuous actions on various protein targets and thus produce non-specific pharmacological effects. To investigate whether the redox reactivity behaves as a critical factor in polyphenol promiscuity, we performed a chemical bioinformatics analysis on the structure-activity relationships of twenty polyphenols. It was found that the gene expression profiles of human cell lines induced by polyphenols were not correlated with the presence or not of redox moieties in the polyphenols, but significantly correlated with their molecular structures. Therefore, it is concluded that the promiscuous actions of polyphenols are likely to result from their inherent structural features rather than their redox potential. PMID:27319142

  3. Molecular Basis of Symbiotic Promiscuity

    PubMed Central

    Perret, Xavier; Staehelin, Christian; Broughton, William J.

    2000-01-01

    Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the “keys” to a succession of legume “doors”. Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys. PMID:10704479

  4. Promiscuous Feminisms for Troubling Times

    ERIC Educational Resources Information Center

    Voithofer, Rick

    2013-01-01

    Looking across the six articles in this issue, this paper argues that promiscuous uses of feminist methodologies offer a unique constellation of conceptual, pragmatic, material, and ethical strategies with which to understand and engage some of the social and cultural tensions that are occurring within and outside schools. It presents a…

  5. Steroid sulfatase gene in XX males.

    PubMed Central

    Mohandas, T K; Stern, H J; Meeker, C A; Passage, M B; Müller, U; Page, D C; Yen, P H; Shapiro, L J

    1990-01-01

    The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:2301402

  6. Sulfatase 1 and sulfatase 2 in hepatocellular carcinoma: associated signaling pathways, tumor phenotypes, and survival.

    PubMed

    Yang, Ju Dong; Sun, Zhifu; Hu, Chunling; Lai, Jinping; Dove, Rebecca; Nakamura, Ikuo; Lee, Ju-Seog; Thorgeirsson, Snorri S; Kang, Koo Jeong; Chu, In-Sun; Roberts, Lewis R

    2011-02-01

    The heparin-degrading endosulfatases sulfatase 1 (SULF1) and sulfatase 2 (SULF2) have opposing effects in hepatocarcinogenesis despite structural similarity. Using mRNA expression arrays, we analyzed the correlations of SULF expression with signaling networks in human hepatocellular carcinomas (HCCs) and the associations of SULF expression with tumor phenotype and patient survival. Data from two mRNA microarray analyses of 139 and 36 HCCs and adjacent tissues were used as training and validation sets. Partek and Metacore software were used to identify SULF correlated genes and their associated signaling pathways. Associations between SULF expression, the hepatoblast subtype of HCC, and survival were examined. Both SULF1 and 2 had strong positive correlations with periostin, IQGAP1, TGFB1, and vimentin and inverse correlations with HNF4A and IQGAP2. Genes correlated with both SULFs were highly associated with the cell adhesion, cytoskeletal remodeling, blood coagulation, TGFB, and Wnt/β-catenin and epithelial mesenchymal transition signaling pathways. Genes uniquely correlated with SULF2 were more associated with neoplastic processes than genes uniquely correlated with SULF1. High SULF expression was associated with the hepatoblast subtype of HCC. There was a bimodal effect of SULF1 expression on prognosis, with patients in the lowest or highest tertile having a worse prognosis than those in the middle tertile. SULFs have complex effects on HCC signaling and patient survival. There are functionally similar associations with cell adhesion, ECM remodeling, TGFB, and WNT pathways, but also unique associations of SULF1 and SULF2. The roles and targeting of the SULFs in cancer require further investigation. PMID:21104785

  7. Steroid Sulfatase Deficiency and Androgen Activation Before and After Puberty

    PubMed Central

    Idkowiak, Jan; Taylor, Angela E.; Subtil, Sandra; O'Neil, Donna M.; Vijzelaar, Raymon; Dias, Renuka P.; Amin, Rakesh; Barrett, Timothy G.; Shackleton, Cedric H. L.; Kirk, Jeremy M. W.; Moss, Celia

    2016-01-01

    Context: Steroid sulfatase (STS) cleaves the sulfate moiety off steroid sulfates, including dehydroepiandrosterone (DHEA) sulfate (DHEAS), the inactive sulfate ester of the adrenal androgen precursor DHEA. Deficient DHEA sulfation, the opposite enzymatic reaction to that catalyzed by STS, results in androgen excess by increased conversion of DHEA to active androgens. STS deficiency (STSD) due to deletions or inactivating mutations in the X-linked STS gene manifests with ichthyosis, but androgen synthesis and metabolism in STSD have not been studied in detail yet. Patients and Methods: We carried out a cross-sectional study in 30 males with STSD (age 6–27 y; 13 prepubertal, 5 peripubertal, and 12 postpubertal) and 38 age-, sex-, and Tanner stage-matched healthy controls. Serum and 24-hour urine steroid metabolome analysis was performed by mass spectrometry and genetic analysis of the STS gene by multiplex ligation-dependent probe amplification and Sanger sequencing. Results: Genetic analysis showed STS mutations in all patients, comprising 27 complete gene deletions, 1 intragenic deletion and 2 missense mutations. STSD patients had apparently normal pubertal development. Serum and 24-hour urinary DHEAS were increased in STSD, whereas serum DHEA and testosterone were decreased. However, total 24-hour urinary androgen excretion was similar to controls, with evidence of increased 5α-reductase activity in STSD. Prepubertal healthy controls showed a marked increase in the serum DHEA to DHEAS ratio that was absent in postpubertal controls and in STSD patients of any pubertal stage. Conclusions: In STSD patients, an increased 5α-reductase activity appears to compensate for a reduced rate of androgen generation by enhancing peripheral androgen activation in affected patients. In healthy controls, we discovered a prepubertal surge in the serum DHEA to DHEAS ratio that was absent in STSD, indicative of physiologically up-regulated STS activity before puberty. This may

  8. Differential Effects of Estrogen Exposure on Arylsulfatase B, Galactose-6-Sulfatase, and Steroid Sulfatase in Rat Prostate Development

    PubMed Central

    Feferman, Leo; Bhattacharyya, Sumit; Birch, Lynn; Prins, Gail S.; Tobacman, Joanne K.

    2014-01-01

    Sulfatase enzymes remove sulfate groups from sulfated steroid hormones, including estrone-sulfate and dehydroepiandrosterone-sulfate, and from sulfated glycosaminoglycans (GAGs), including chondroitin sulfates and heparan sulfate. The enzymes N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) and N-acetylgalactosamine-6-sulfatase (GALNS), which remove sulfate groups from the sulfated GAGs chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate, respectively, have not been studied in prostate development previously. In this report, the endogenous variation and the impact of exogenous estradiol benzoate on the immunohistochemistry and activity of ARSB and GALNS in post-natal (days 1–30) ventral rat prostate are presented, as well as measurements of steroid sulfatase activity (STS), C4S, total sulfated GAGs, and versican, an extracellular matrix proteoglycan with chondroitin sulfate attachments on days 5 and 30. Findings demonstrate distinct and reciprocal localization of ARSB and GALNS, with ARSB predominant in the stroma and GALNS predominant in the epithelium. Control ARSB activity increased significantly between days 5 and 30, but following estrogen exposure (estradiol benzoate 25 µg in 25 µl sesame oil subcutaneously on days 1, 3, and 5), activity was reduced and the observed increase on day 30 was inhibited. However, estrogen treatment did not inhibit the increase in GALNS activity between days 5 and 30, and reduced STS activity by 50% on both days 5 and 30 compared to vehicle control. Sulfated GAGs, C4S, and the extracellular matrix proteoglycan versican declined between days 5 and 30 in the control, but these declines were inhibited following estrogen. Study findings indicate distinct variation in expression and activity of sulfatases, sulfated GAGs, C4S, and versican in the process of normal prostate development, and disruption of these events by exogenous estrogen. PMID:24508597

  9. Enzyme promiscuity in earthworm serine protease: substrate versatility and therapeutic potential.

    PubMed

    Verma, Mahendra Kumar; Pulicherla, K K

    2016-04-01

    Enzymes are the most versatile molecules in the biological world. These amazing molecules play an integral role in the regulation of various metabolic pathways and physiology subsequently. Promiscuity of an enzyme is the capacity to catalyze additional biochemical reactions besides their native one. Catalytic promiscuity has shown great impact in enzyme engineering for commercial enzyme and therapeutics with natural or engineered catalytic promiscuity. The earthworm serine protease (ESP) is a classic example of enzyme promiscuity and studied for its therapeutic potential over the last few decades. The ESP was reported for several therapeutic properties and fibrinolytic activity has been much explored. ESP, a complex enzyme exists as several isoforms of molecular weight ranging from 14 to 33 kDa. The fibrinolytic capacity of the enzyme has been studied in different species of earthworm and molecular mechanism is quite different from conventional thrombolytics. Cytotoxic and anti-tumor activities of ESP were evaluated using several cancer cell lines. Enzyme had shown tremendous scope in fighting against plant viruses and microbes. ESP is also reported for anti-inflammatory activity and anti-oxidant property. Apart from these, recently, ESP is reported for DNase activity. The daunting challenge for researchers is to understand the molecular mechanism for such diverse properties and possibility of enzyme promiscuity. This review emphasizes molecular mechanism of ESP governing various biochemical reactions. Further, the concept of enzyme promiscuity in ESP towards development of novel enzyme based drugs has been reviewed in this study. PMID:26739820

  10. Level of Education, Sexual Promiscuity, and AIDS.

    ERIC Educational Resources Information Center

    Krull, Catherine D.

    1994-01-01

    Responses of 595 unmarried persons on the 1991 General Social Survey suggest that educational attainment indirectly increases promiscuous sexual behavior by liberalizing sexual attitudes. In a second study, the incidence of AIDS was partly explained by educational attainment, liberal sexual attitudes, and promiscuous sexual practices. (KS)

  11. Nosology, ontology and promiscuous realism.

    PubMed

    Binney, Nicholas

    2015-06-01

    Medics may consider worrying about their metaphysics and ontology to be a waste of time. I will argue here that this is not the case. Promiscuous realism is a metaphysical position which holds that multiple, equally valid, classification schemes should be applied to objects (such as patients) to capture different aspects of their complex and heterogeneous nature. As medics at the bedside may need to capture different aspects of their patients' problems, they may need to use multiple classification schemes (multiple nosologies), and thus consider adopting a different metaphysics to the one commonly in use. PMID:25389077

  12. Sulfatase inhibitors for recidivist breast cancer treatment: A chemical review.

    PubMed

    Shah, Ramanpreet; Singh, Jatinder; Singh, Dhandeep; Jaggi, Amteshwar Singh; Singh, Nirmal

    2016-05-23

    Steroid sulfatase (STS) plays a momentous role in the conversion of sulfated steroids, which are biologically inactive, into biologically active un-sulfated steroid hormones, which support the development and growth of a number of hormone-dependent cancers, including breast cancer. Therefore, inhibitors of STS are supposed to be potential drugs for the treatment of breast and other steroid-dependent cancers. The present review concentrates on broad chemical classification of steroid sulfatase inhibitors. The inhibitors reviewed are classified into four main categories: Steroid sulfamate based inhibitors; Steroid non-sulfamate based inhibitors; Non-steroidal sulfamate based inhibitors; Non-steroidal non-sulfamate based inhibitors. A succinct overview of current treatment of cancer, estradiol precursors, STS enzyme and its role in breast cancer is herein described. PMID:26974384

  13. Multiple sulfatase deficiency is due to hypomorphic mutations of the SUMF1 gene.

    PubMed

    Annunziata, Ida; Bouchè, Valentina; Lombardi, Alessia; Settembre, Carmine; Ballabio, Andrea

    2007-09-01

    Sulfatases catalyze the hydrolysis of sulfate ester bonds from a wide variety of substrates and are implicated in several human inherited diseases. Multiple sulfatase deficiency (MSD) is a rare autosomal recessive disorder characterized by the simultaneous deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the alpha-formylglycine generating enzyme (FGE), which is responsible for the post-translational modification of sulfatases. In all MSD patients, residual sulfatase activities are detectable, at variable levels. To correlate the nature of the residual sulfatase activities detected in MSD patients with residual FGE activity, four FGE mutants (i.e. p.S155P, p.R224W, p.R345C, p.R349W) found in homozygosis in MSD patients were analyzed. Using viral-mediated gene delivery, these mutants were over-expressed in mouse embryonic fibroblasts (MEFs) from a recently developed Sumf1 KO mouse line which is completely devoid of all sulfatase activities. The results obtained indicate that mutant SUMF1 cDNAs encode stable SUMF1 proteins which are of the appropriate molecular weight and are properly localized in the endoplasmic reticulum. Expression of these cDNAs in Sumf1-/- MEFs results in partial rescue of sulfatase activities. These data indicate that MSD is due to hypomorphic SUMF1 mutations and suggest that complete loss of SUMF1 function is likely to be lethal in humans. PMID:17657823

  14. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25500069

  15. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  16. Modeling catalytic promiscuity in the alkaline phosphatase superfamily

    PubMed Central

    Duarte, Fernanda; Amrein, Beat Anton

    2013-01-01

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein

  17. Sequence correlations shape protein promiscuity

    NASA Astrophysics Data System (ADS)

    Lukatsky, David B.; Afek, Ariel; Shakhnovich, Eugene I.

    2011-08-01

    We predict analytically that diagonal correlations of amino acid positions within protein sequences statistically enhance protein propensity for nonspecific binding. We use the term "promiscuity" to describe such nonspecific binding. Diagonal correlations represent statistically significant repeats of sequence patterns where amino acids of the same type are clustered together. The predicted effect is qualitatively robust with respect to the form of the microscopic interaction potentials and the average amino acid composition. Our analytical results provide an explanation for the enhanced diagonal correlations observed in hubs of eukaryotic organismal proteomes [J. Mol. Biol. 409, 439 (2011)], 10.1016/j.jmb.2011.03.056. We suggest experiments that will allow direct testing of the predicted effect.

  18. PubChem promiscuity: a web resource for gathering compound promiscuity data from PubChem

    PubMed Central

    Canny, Stephanie A.; Cruz, Yasel; Southern, Mark R.; Griffin, Patrick R.

    2012-01-01

    Summary: Promiscuity counts allow for a better understanding of a compound's assay activity profile and drug potential. Although PubChem contains a vast amount of compound and assay data, it currently does not have a convenient or efficient method to obtain in-depth promiscuity counts for compounds. PubChem promiscuity fills this gap. It is a Java servlet that uses NCBI Entrez (eUtils) web services to interact with PubChem and provide promiscuity counts in a variety of categories along with compound descriptors, including PAINS-based functional group detection. Availability: http://chemutils.florida.scripps.edu/pcpromiscuity Contact: southern@scripps.edu PMID:22084255

  19. Galactokinase promiscuity: a question of flexibility?

    PubMed

    McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

    2016-02-01

    Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. PMID:26862196

  20. Functional Promiscuity of the COG0720 Family

    PubMed Central

    Phillips, Gabriela; Grochowski, Laura L.; Bonnett, Shilah; Xu, Huimin; Bailly, Marc; Haas-Blaby, Crysten; El Yacoubi, Basma; Iwata-Reuyl, Dirk; White, Robert H.; de Crécy-Lagard, Valérie

    2011-01-01

    The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function. PMID:21999246

  1. Determining the Degree of Promiscuity of Extensively Assayed Compounds

    PubMed Central

    Jasial, Swarit; Hu, Ye; Bajorath, Jürgen

    2016-01-01

    In the context of polypharmacology, an emerging concept in drug discovery, promiscuity is rationalized as the ability of compounds to specifically interact with multiple targets. Promiscuity of drugs and bioactive compounds has thus far been analyzed computationally on the basis of activity annotations, without taking assay frequencies or inactivity records into account. Most recent estimates have indicated that bioactive compounds interact on average with only one to two targets, whereas drugs interact with six or more. In this study, we have further extended promiscuity analysis by identifying the most extensively assayed public domain compounds and systematically determining their promiscuity. These compounds were tested in hundreds of assays against hundreds of targets. In our analysis, assay promiscuity was distinguished from target promiscuity and separately analyzed for primary and confirmatory assays. Differences between the degree of assay and target promiscuity were surprisingly small and average and median degrees of target promiscuity of 2.6 to 3.4 and 2.0 were determined, respectively. Thus, target promiscuity remained at a low level even for most extensively tested active compounds. These findings provide further evidence that bioactive compounds are less promiscuous than drugs and have implications for pharmaceutical research. In addition to a possible explanation that drugs are more extensively tested for additional targets, the results would also support a “promiscuity enrichment model” according to which promiscuous compounds might be preferentially selected for therapeutic efficacy during clinical evaluation to ultimately become drugs. PMID:27082988

  2. Cerebroside Sulfatase Activity in Cultivated Human Skin Fibroblasts and Amniotic Fluid Cells

    ERIC Educational Resources Information Center

    Booth, Carol W.; And Others

    1975-01-01

    Prenatal monitoring for metachromatic leukodystrophy (a fatal inherited metabolic disorder) suggested that the determination of levels of cerebroside sulfatase in the amniotic fluid helped in the prenatal detection of this disorder. (DB)

  3. Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent.

    PubMed

    Sinharay, Sanhita; Fernández-Cuervo, Gabriela; Acfalle, Jasmine P; Pagel, Mark D

    2016-05-01

    A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity. PMID:26956002

  4. Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

    PubMed Central

    Bloom, Jesse D; Romero, Philip A; Lu, Zhongyi; Arnold, Frances H

    2007-01-01

    Background Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's original biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution. Results Here we investigate this possibility experimentally by examining how cytochrome P450 enzymes that have evolved neutrally with respect to activity on a single substrate have changed in their abilities to catalyze reactions on five other substrates. We find that the enzymes have sometimes changed as much as four-fold in the promiscuous activities. The changes in promiscuous activities tend to increase with the number of mutations, and can be largely rationalized in terms of the chemical structures of the substrates. The activities on chemically similar substrates tend to change in a coordinated fashion, potentially providing a route for systematically predicting the change in one activity based on the measurement of several others. Conclusion Our work suggests that initially neutral genetic drift can lead to substantial changes in protein functions that are not currently under selection, in effect poising the proteins to more readily undergo functional evolution should selection favor new functions in the future. Reviewers This article was reviewed by Martijn Huynen, Fyodor Kondrashov, and Dan Tawfik (nominated by Christoph Adami). PMID:17598905

  5. No genome barriers to promiscuous DNA

    NASA Astrophysics Data System (ADS)

    Lewin, R.

    1984-06-01

    Farrelly and Butow (1983) used the term 'promiscuous DNA' in their report of the apparent natural transfer of yeast mitochondrial DNA sequences into the nuclear genome. Ellis (1982) applied the same term in an editorial comment. It is pointed out since that time the subject of DNA's promiscuity has exploded with a series of reports. According to a report by Stern (1984), movement of DNA sequences between chloroplasts and mitochondria is not just a rare event but is a rampant process. It was recently concluded that 'the widespread presence of ctDNA sequences in plant mtDNA is best regarded as a dramatic demonstration of the dynamo nature of interactions between the chloroplast and the mitochondrion, similar to the ongoing process of interorganellar DNA transfer already documented between mitochondrion and nucleus and between chloroplast and nucleus'.

  6. Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

    PubMed Central

    Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

    2016-01-01

    Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

  7. Steroid Sulfatase Inhibitors Based on Phosphate and Thiophosphate Flavone Analogs.

    PubMed

    Kozak, Witold; Daśko, Mateusz; Masłyk, Maciej; Kubiński, Konrad; Rachon, Janusz; Demkowicz, Sebastian

    2015-12-01

    A series of phosphate and thiophosphate flavone derivatives were synthesized and biologically evaluated in vitro for inhibition of steroid sulfatase (STS) activity. The described synthesis includes the straightforward preparation of 7-hydroxy-2-phenyl-4H-chromen-4-one 3a, 2-(4-fluorophenyl)-7-hydroxy-4H-chromen-4-one 3b, 7-hydroxy-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one 3c, 7-hydroxy-2-(p-tolyl)-4H-chromen-4-one 3d modified with different phosphate or thiophosphate moieties. The inhibitory properties of the synthesized compounds were tested against human placenta STS. Some of the novel STS inhibitors had good activities against STS. In particular, the bis-(4-oxo-2-(p-tolyl)-4H-chromen-7-yl) hydrogenthiophosphate, 6i had the most potent inhibitory effect with an IC50 value of 3.25 µM as compared to an IC50 value of 8.50 µM for the 2-(4-trifluoromethylphenyl)-chromen-4-one-7-O-sulfamate used as a reference. PMID:26415657

  8. [Proximity, intimacy and promiscuity in care].

    PubMed

    Flicourt, Nadia

    2015-04-01

    Lying at the heart of the intimacy of the other person, the nature of care supposes that the caregiver identifies the components resulting from the proximity and the invasion of the patient's personal space, where perceptions and representations give rise to reactive emotions and behaviour. Between modesty and nudity, proximity and promiscuity, caregivers have to adjust their approach of proper care, limiting the risks of intrusion. PMID:26043630

  9. A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme.

    PubMed

    Roeser, Dirk; Preusser-Kunze, Andrea; Schmidt, Bernhard; Gasow, Kathrin; Wittmann, Julia G; Dierks, Thomas; von Figura, Kurt; Rudolph, Markus Georg

    2006-01-01

    The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism. PMID:16368756

  10. Statistically enhanced promiscuity of structurally correlated patterns

    NASA Astrophysics Data System (ADS)

    Lukatsky, D. B.; Shakhnovich, E. I.

    2008-02-01

    We predict that patterns with correlated surface density of atoms have statistically higher promiscuity (ability to bind stronger to an arbitrary pattern) as compared with noncorrelated patterns with the same average surface density. We suggest that this constitutes a generic design principle for highly connected proteins (hubs) in protein interaction networks. We develop an analytical theory for this effect. We show that our key predictions are generic and independent, qualitatively, on the specific form of the interatomic interaction potential, provided it has a finite range.

  11. SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Ennemann, Eva Charlotte; Radhakrishnan, Karthikeyan; Schmidt, Bernhard; Chakrapani, Anupam; Christen, Hans-Jürgen; Moser, Hugo; Steinmann, Beat; Dierks, Thomas; Gärtner, Jutta

    2011-03-01

    Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE. PMID:21224894

  12. Location of Aryl Sulfatase in Conidia and Young Mycelia of Neurospora crassa

    PubMed Central

    Scott, Walter A.; Metzenberg, Robert L.

    1970-01-01

    Aryl sulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was found to have multiple locations in Neurospora conidia. Some enzyme activity remained in the supernatant when a spore suspension was centrifuged or filtered. Part of the cell-bound activity could be detected by adding the assay ingredients to a suspension of intact spores (patent enzyme), and additional activity was only detectable when the spores were first treated to destroy their permeability barriers (cryptic enzyme). Such treatments include: disruption with an X-press, brief rinsing with chloroform or acetone, incubation at 60 C for 5 min, and incubation with phenethyl alcohol, nystatin, or ascosin. Part of the patent aryl sulfatase was inactivated by briefly acid treating the intact spores (no loss of conidial viability). This enzyme was considered to have a cell surface location. Some enzyme was acid-resistant in intact spores, but all of the enzyme was acid-sensitive in spores whose permeability barriers had been disrupted. The pH dependence, kinetic properties, and p-nitrophenyl sulfate uptake were investigated in acid-treated conidia. No aryl sulfatase was detected in ascospores. Young mycelia contained more aryl sulfatase than did conidia, but little, if any, was secreted into the growth medium. Cryptic activity was demonstrated in young mycelia by brief chloroform treatment or by rinsing the cells with 0.1 m acetate buffer. Enzyme activity in young mycelia was completely labile to acid treatment, as was cell viability. PMID:16559101

  13. Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications.

    PubMed

    Pasqualini, J R; Chetrite, G S

    1999-01-01

    Estrone sulfate (E1S) is concentrated in high levels in human breast cancer tissue. The values are particularly high in postmenopausal women and many times those circulating in the plasma. Also, the tissular concentration of this conjugate are significantly higher in tumoural tissue than in the area of the breast considered as normal. The enzyme which hydrolyzes E1S: sulfatase, as well as the enzyme which biosynthesises this conjugate: sulfotransferase, are present in significant concentrations in breast cancer tissue. Consequently, E1S is a balance between the activities of the two enzymes. As breast cancer tissue has all the enzymes necessary for the synthesis of estradiol (E2), and the formation of E2 from E1S 'via sulfatase' is the main pathway, it was very attractive to explore inhibitory agents of this enzyme. It was observed that different substances including antiestrogens (4-hydroxytamoxifen, ICI 164,384) and various progestins (promegestone, nomegestrol acetate, medrogestone) as well as Org OD14 (tibolone) can block the sulfatase activity. In addition, it was demonstrated that different progestins (medrogestone, nomegestrol acetate, TX-525) and org OD14 can stimulate the sulfotransferase activity for the formation of the biologically inactive E1S. It is concluded that the inhibition of sulfatase and the stimulation of sulfotransferase activity can open interesting possibilities to explore these effects in patients with breast cancer. PMID:10419004

  14. Control of sulfatase activity by nomegestrol acetate in normal and cancerous human breast tissues.

    PubMed

    Chetrite, Gérard Samuel; Thomas, Jean-Louis; Shields-Botella, Jaqueline; Cortes-Prieto, Joaquin; Philippe, Jean-Claude; Pasqualini, Jorge Raul

    2005-01-01

    Nomegestrol acetate (NOMAC), a 17alpha-hydroxy-nor-progesterone derivative (17alpha-acetoxy-6-methyl-19-nor-4,6-pregnadiene-3,20-dione, the active substance in Lutenyl), is a potent and useful clinical synthetic progestin for the treatment of menopausal complaints and is under current development for oral contraception. Previous studies in this laboratory demonstrated that NOMAC can block sulfatase and 17beta-hydroxysteroid dehydrogenase, the enzymes involved in the biosynthesis and transformation of estradiol (E2) in hormone-dependent MCF-7 and T-47D breast cancer cells. In the present study, the effect of NOMAC on sulfatase activity using total breast cancer tissue, compared to the effect in normal breast tissue, was explored. Slices of tumoral or normal breast tissues (45-65 mg) were incubated in buffer (20 mM Tris-HCl, pH 7.2) with physiological concentrations of [3H]-estrone sulfate (5x10(-9) M), alone or in the presence of nomegestrol acetate (5x10(-5) - 5x10(-7) - 5x10(-9) M), for 4 h at 37 degrees C. Estrone sulfate (E1S), estrone (E1) and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [3H]- E1S was only converted to [3H]- E1 and not to [3H]- E2, in normal or cancerous breast tissues, which suggests a low or no 17beta-HSD activity under these experimental conditions. The sulfatase activity was more intense with breast cancer tissue than normal tissue, since the concentrations of E1 were 42.5 +/- 3.4 and 27.2 +/- 2.5 pg/mg tissue, respectively. NOMAC, at the concentration of 5x10(-5) M, inhibited this conversion by 49.2% and 40.8% in cancerous and normal breast tissues, respectively. The sulfatase inhibition at low concentration (5x10(-7) M) was 32.5% and 22.8%, respectively. It is concluded that sulfatase activity is almost twice as potent in cancerous breast tissues than in normal tissues. Nomegestrol acetate is a strong anti-sulfatase agent, in particular with cancerous breast

  15. Discovery of a novel iota carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora

    PubMed Central

    Genicot, Sabine M.; Groisillier, Agnès; Rogniaux, Hélène; Meslet-Cladière, Laurence; Barbeyron, Tristan; Helbert, William

    2014-01-01

    Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of carrageenans. As a result of the screening, an iota-carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain PscT. It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc ι-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH ~8.3 and at 40 ± 5°C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc ι-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-carrageenan. The purified enzyme could transform iota-carrageenan into hybrid iota-/alpha- or pure alpha-carrageenan under controlled conditions. The gene encoding Psc ι-CgsA, a protein of 1038 amino acids, was cloned into Escherichia coli, and the sequence analysis revealed that Psc ι-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P. carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes. PMID:25207269

  16. Discovery of a novel iota carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora

    NASA Astrophysics Data System (ADS)

    Genicot, Sabine; Groisillier, Agnès; Rogniaux, Hélène; Meslet-Cladière, Laurence; Barbeyron, Tristan; Helbert, William

    2014-08-01

    Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of carrageenans. As a result of the screening, an iota-carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain PscT. It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc ?-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH ~8.3 and at 40°C ± 5°C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc ?-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-carrageenan. The purified enzyme could transform iota-carrageenan into hybrid iota-/alpha- or pure alpha-carrageenan under controlled conditions. The gene encoding Psc ?-CgsA, a protein of 1038 amino acids, was cloned into Escherichia coli, and the sequence analysis revealed that Psc ?-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P. carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes.

  17. A Promiscuous De Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.

    PubMed

    Garrabou, Xavier; Beck, Tobias; Hilvert, Donald

    2015-05-01

    Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution. PMID:25777153

  18. Multisite Promiscuity in the Processing of Endogenous Substrates By Human Carboxylesterase 1

    SciTech Connect

    Bencharit, S.; Edwards, C.C.; Morton, C.L.; Howard-Williams, E.L.; Kuhn, P.; Potter, P.M.; Redinbo, M.R.; /North Carolina U. /St. Jude Children's Hosp., Memphis /SLAC, SSRL

    2007-01-16

    Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.

  19. A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

    PubMed Central

    Galperin, M. Y.; Bairoch, A.; Koonin, E. V.

    1998-01-01

    Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy. PMID:10082381

  20. The Promiscuous Nature of Stars in Clusters

    NASA Astrophysics Data System (ADS)

    Hurley, Jarrod R.; Shara, Michael M.

    2002-05-01

    The recent availability of special-purpose computers designed for calculating gravitational interactions of N bodies at extremely high speed has provided the means to model globular clusters on a star-by-star basis for the first time. By endeavoring to make the N-body codes that operate on these machines as realistic as possible, the addition of stellar evolution being one example, we are learning much about the interaction between the star cluster itself and the stars it contains. A fascinating aspect of this research is the ability to follow the orbits of individual stars in detail and to document the formation of observed exotic systems. This has revealed that many stars within a star cluster lead wildly promiscuous lives, interacting often intimately and in rapid succession with a variety of neighbors.

  1. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  2. Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily.

    PubMed

    Pabis, Anna; Kamerlin, Shina Caroline Lynn

    2016-04-01

    Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity. PMID:26716576

  3. Sexual promiscuity: knowledge of dangers in institutions of higher learning.

    PubMed

    Ebong, R D

    1994-06-01

    Knowledge of dangers of sexual promiscuity was assessed in 2 institutions of higher learning. The objectives were to find out the knowledge of medical and social consequences as well as the factors responsible for sexual promiscuity among Nigerian youths. The study also assessed the discrepancies in societal concept of sex norms for males and females. The result was used as an index to determine the need for sex education for Nigerian youths. A total of 200 students (100 from each school) was assessed by random selection and use of a questionnaire. The result showed that students had a fair knowledge of sexual promiscuity, although in terms of medical consequences the knowledge was low for both groups. On social consequences, the knowledge was fair for both groups. Students agreed that lack of financial support and of supervision from parents and teachers were among the causes of sexual promiscuity. Recommendations were made for Health Education in these areas in institutions of higher learning. Also, recommendations were made for parental education on how to bring up, and care for, their adolescents to reduce the problems of sexual promiscuity. It was also recommended that a compulsory course on sexual promiscuity should be included in the syllabus in institutions of higher learning. PMID:7932483

  4. Is Promiscuous CALB a Good Scaffold for Designing New Epoxidases?

    PubMed

    Bordes, Isabel; Recatalá, José; Świderek, Katarzyna; Moliner, Vicent

    2015-01-01

    Candida Antarctica lipase B (CALB) is a well-known enzyme, especially because of its promiscuous activity. Due to its properties, CALB was widely used as a benchmark for designing new catalysts for important organic reactions. The active site of CALB is very similar to that of soluble epoxide hydrolase (sEH) formed by a nucleophile-histidine-acid catalytic triad and an oxyanion hole typical for molecular structures derived from processes of α/β hydrolases. In this work we are exploring these similarities and proposing a Ser105Asp variant of CALB as a new catalyst for epoxide hydrolysis. In particular, the hydrolysis of the trans-diphenylpropene oxide (t-DPPO) is studied by means of quantum cluster models mimicking the active site of both enzymes. Our results, based on semi-empirical and DFT calculations, suggest that mutant Ser105Asp CALB is a good protein scaffold to be used for the bio-synthesis of chiral compounds. PMID:26404218

  5. Steroid sulfatase-deficient mice exhibit endophenotypes relevant to Attention Deficit Hyperactivity Disorder

    PubMed Central

    Trent, Simon; Dennehy, Alison; Richardson, Heather; Ojarikre, Obah A.; Burgoyne, Paul S.; Humby, Trevor; Davies, William

    2012-01-01

    Summary Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental condition characterised by inattention, impulsivity and hyperactivity; it is frequently co-morbid with anxiety and conduct disorders, sleep perturbation and abnormal consummatory behaviours. Recent studies have implicated the neurosteroid-modulating enzyme steroid sulfatase (STS) as a modulator of ADHD-related endophenotypes. The effects of steroid sulfatase deficiency on homecage activity, feeding/drinking behaviours, anxiety-related behaviours (assayed in light-dark box and open field paradigms), social dominance and serum steroid hormone levels were determined by comparing 40,XY and 39,XY*O mice. Subsequently, mice administered the steroid sulfatase inhibitor COUMATE acutely were compared to vehicle-treated mice on behavioural tasks sensitive to enzyme deficiency to dissociate between its developmental and ongoing effects. 39,XY*O mice exhibited heightened reactivity to a novel environment, hyperactivity in the active phase, and increased water (but not food) consumption relative to 40,XY mice during a 24 h period; the former group also demonstrated evidence for heightened emotional reactivity. There was no difference in social dominance between the 40,XY and 39,XY*O mice. COUMATE administration had no effect on homecage activity, water consumption or anxiety measures in the open field. 39,XY*O mice exhibited significantly lower dehydroepiandrosterone (DHEA) serum levels than 40,XY mice, but equivalent corticosterone levels. Together with previous data, the present results suggest that steroid sulfatase may influence core and associated ADHD behavioural endophenotypes via both developmental and ongoing mechanisms, and that the 39,XY*O model may represent a useful tool for elucidating the neurobiological basis of these endophenotypes. PMID:21723668

  6. Genetic and Pharmacological Modulation of the Steroid Sulfatase Axis Improves Response Control; Comparison with Drugs Used in ADHD

    PubMed Central

    Davies, William; Humby, Trevor; Trent, Simon; Eddy, Jessica B; Ojarikre, Obah A; Wilkinson, Lawrence S

    2014-01-01

    Maladaptive response control is a feature of many neuropsychiatric conditions, including attention deficit hyperactivity disorder (ADHD). As ADHD is more commonly diagnosed in males than females, a pathogenic role for sex-linked genes has been suggested. Deletion or point mutation of the X-linked STS gene, encoding the enzyme steroid sulfatase (STS) influences risk for ADHD. We examined whether deletion of the Sts gene in the 39,XY*O mouse model, or pharmacological manipulation of the STS axis, via administration of the enzyme substrate dehydroepiandrosterone sulfate or the enzyme inhibitor COUMATE, influenced behavior in a novel murine analog of the stop-signal reaction time task used to detect inhibitory deficits in individuals with ADHD. Unexpectedly, both the genetic and pharmacological treatments resulted in enhanced response control, manifest as highly specific effects in the ability to cancel a prepotent action. For all three manipulations, the effect size was comparable to that seen with the commonly used ADHD therapeutics methylphenidate and atomoxetine. Hence, converging genetic and pharmacological evidence indicates that the STS axis is involved in inhibitory processes and can be manipulated to give rise to improvements in response control. While the precise neurobiological mechanism(s) underlying the effects remain to be established, there is the potential for exploiting this pathway in the treatment of disorders where failures in behavioral inhibition are prominent. PMID:24842408

  7. Finding promiscuous old drugs for new uses.

    PubMed

    Ekins, Sean; Williams, Antony J

    2011-08-01

    From research published in the last six years we have identified 34 studies that have screened libraries of FDA-approved drugs against various whole cell or target assays. These studies have each identified one or more compounds with a suggested new bioactivity that had not been described previously. We now show that 13 of these drugs were active against more than one additional disease, thereby suggesting a degree of promiscuity. We also show that following compilation of all the studies, 109 molecules were identified by screening in vitro. These molecules appear to be statistically more hydrophobic with a higher molecular weight and AlogP than orphan-designated products with at least one marketing approval for a common disease indication or one marketing approval for a rare disease from the FDA's rare disease research database. Capturing these in vitro data on old drugs for new uses will be important for potential reuse and analysis by others to repurpose or reposition these or other existing drugs. We have created databases which can be searched by the public and envisage that these can be updated as more studies are published. PMID:21607776

  8. Sanfilippo D syndrome: Estimation of N-acetylglucosamine-6-sulfatase activity with a radiolabeled monosulfated disaccharide substrate

    SciTech Connect

    Freeman, C.; Hopwood, J.J.

    1989-02-01

    N-Acetylglucosamine-6-sulfatase activity was assayed by incubation of the radiolabeled disaccharide O-(a-N-acetylglucosamine-6-sulfate)-(1----3)-L-(6-/sup 3/H)-idonic acid (GlcNAc6S-IdOA), with homogenates of leucocytes, cultured fibroblasts, and urine from normal individuals, patients affected with N-acetylglucosamine-6-sulfatase-deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses and lysosomal storage disorders. The assay clearly distinguished affected homozygotes from their obligate heterozygotes and normal controls and other lysosomal storage disorders. Sulfatase activity in fibroblasts, leucocytes, and urine toward GlcNAc6S-IdOA exhibited a pH optimum at 4.2, 4.5, and 5.1, respectively. Sulfatase activity in fibroblasts had an apparent Km of 7.2 microM and was significantly inhibited by both sulfate and phosphate ions. The action of fibroblast or leucocyte N-acetylglucosamine-6-sulfatase activity toward GlcNAc6S-IdOA is recommended for the routine enzymatic detection and classification of mucopolysaccharidosis type IIID patients.

  9. Evolving new protein-protein interaction specificity through promiscuous intermediates.

    PubMed

    Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N; Perchuk, Barrett S; Crosson, Sean; Laub, Michael T

    2015-10-22

    Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution. PMID:26478181

  10. Promiscuity and the evolution of sexual transmitted diseases

    NASA Astrophysics Data System (ADS)

    Gonçalves, Sebastián; Kuperman, Marcelo; Ferreira da Costa Gomes, Marcelo

    2003-09-01

    We study the relation between different social behaviors and the onset of epidemics in a model for the dynamics of sexual transmitted diseases. The model considers the society as a system of individual sexuated agents that can be organized in couples and interact with each other. The different social behaviors are incorporated assigning what we call a promiscuity value to each individual agent. The individual promiscuity is taken from a distribution and represents the daily probability of going out to look for a sexual partner, abandoning its eventual mate. In terms of this parameter we find a threshold for the epidemic which is much lower than the classical SIR model prediction, i.e., R0 (basic reproductive number)=1. Different forms for the distribution of the population promiscuity are considered showing that the threshold is weakly sensitive to them. We study the homosexual and the heterosexual case as well.

  11. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

  12. Promiscuity and selectivity of bitter molecules and their receptors.

    PubMed

    Di Pizio, Antonella; Niv, Masha Y

    2015-07-15

    Bitter taste is essential for survival, as it protects against consuming poisonous compounds, which are often bitter. Bitter taste perception is mediated by bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs). The number of TAS2R subtypes is species-dependent, and varies from 3 in chicken to 50 in frog. TAS2Rs present an intriguing case for studying promiscuity: some of the receptors are still orphan, or have few known agonists, while others can be activated by numerous, structurally dissimilar compounds. The ligands also vary in the repertoire of TAS2Rs that they activate: some bitter compounds are selective toward a single TAS2R, while others activate multiple TAS2Rs. Selectivity/promiscuity profile of bitter taste receptors and their compounds was explored by a chemoinformatic approach. TAS2R-promiscuous and TAS2R-selective bitter molecules were found to differ in chemical features, such as AlogP, E-state, total charge, number of rings, globularity, and heavy atom count. This allowed the prediction of bitter ligand selectivity toward TAS2Rs. Interestingly, while promiscuous TAS2Rs are activated by both TAS2R-promiscuous and TAS2R-selective compounds, almost all selective TAS2Rs in human are activated by promiscuous compounds, which are recognized by other TAS2Rs anyway. Thus, unique ligands, that may have been the evolutionary driving force for development of selective TAS2Rs, still need to be unraveled. PMID:25934224

  13. Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.

    PubMed

    Dierks, Thomas; Schmidt, Bernhard; Borissenko, Ljudmila V; Peng, Jianhe; Preusser, Andrea; Mariappan, Malaiyalam; von Figura, Kurt

    2003-05-16

    C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure. PMID:12757705

  14. Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme.

    PubMed

    Dierks, Thomas; Dickmanns, Achim; Preusser-Kunze, Andrea; Schmidt, Bernhard; Mariappan, Malaiyalam; von Figura, Kurt; Ficner, Ralf; Rudolph, Markus Georg

    2005-05-20

    Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate. PMID:15907468

  15. Harnessing natural product assembly lines: structure, promiscuity, and engineering.

    PubMed

    Ladner, Christopher C; Williams, Gavin J

    2016-03-01

    Many therapeutically relevant natural products are biosynthesized by the action of giant mega-enzyme assembly lines. By leveraging the specificity, promiscuity, and modularity of assembly lines, a variety of strategies has been developed that enables the biosynthesis of modified natural products. This review briefly summarizes recent structural advances related to natural product assembly lines, discusses chemical approaches to probing assembly line structures in the absence of traditional biophysical data, and surveys efforts that harness the inherent or engineered promiscuity of assembly lines for the synthesis of non-natural polyketides and non-ribosomal peptide analogues. PMID:26527577

  16. Evidence that the Bacteroides thetaiotaomicron chondroitin lyase II gene is adjacent to the chondro-4-sulfatase gene and may be part of the same operon.

    PubMed Central

    Guthrie, E P; Salyers, A A

    1987-01-01

    The chondroitin lyase II gene from Bacteroides thetaiotaomicron has previously been cloned in Escherichia coli on a 7.8-kilobase (kb) fragment (pA818). In E. coli, the chondroitin lyase II gene appeared to be expressed from a promoter that was about 0.5 kb from the beginning of the gene. However, when a subcloned 5-kb fragment from pA818 which contained the chondroitin lyase II gene and the promoter from which the gene is expressed in E. coli was introduced into B. thetaiotaomicron on a multicopy plasmid (pEG800), the chondroitin lyase specific activity of B. thetaiotaomicron was not altered. Further evidence that the promoter that is recognized in E. coli may not be the promoter from which the chondroitin lyase II gene is transcribed in B. thetaiotaomicron was obtained by making an insertion in the B. thetaiotaomicron chromosome at a point which is 1 kb upstream from the chondroitin lyase II gene. This insertion stopped synthesis of the chondroitin lyase II gene product, as would be predicted if the gene was part of an operon and was transcribed in B. thetaiotaomicron from a promoter that was at least 1 kb upstream from the chondroitin lyase II gene. A region of pA818 which was adjacent to the chondroitin lyase II gene and which included the region used to make the insertional mutation was found to code for chondro-4-sulfatase, an enzyme that breaks down one of the products of the chondroitin lyase reaction. The upstream insertion mutant of B. thetaiotaomicron which stopped synthesis of chondroitin lyase II had no detectable chondro-4-sulfatase activity. This mutant was still able to grow on chondroitin sulfate, although the rate of growth was slower than that of the wild type. Images PMID:3029024

  17. Much More than Power: The Pedagogy of Promiscuous Black Feminism

    ERIC Educational Resources Information Center

    Huckaby, M. Francyne

    2013-01-01

    This paper explores promiscuous black feminism by juxtaposing black feminism, Foucualt's poststructuralism, and my grandmother. The tensions created by these juxtapositions illuminate the ways black feminism and poststructuralism are resources and challenges to each other, and how both offer understandings of the relations at play that shape…

  18. Promiscuity in mice is associated with increased vaginal bacterial diversity

    NASA Astrophysics Data System (ADS)

    Macmanes, Matthew David

    2011-11-01

    Differences in the number of sexual partners (i.e., mating system) have the potential to exert a strong influence on the bacterial communities present in reproductive structures like the vagina. Because this structure serves as a conduit for gametes, bacteria present there may have a pronounced, direct effect on host reproductive success. As a first step towards the identification of the relationship between sexual behavior and potentially pathogenic bacterial communities inhabiting vital reproductive structures, as well as their potential effects on fitness, I sought to quantify differences in bacterial diversity in a promiscuous and monogamous mammal species. To accomplish this, I used two sympatric species of Peromyscus rodents— Peromyscus californicus and Peromyscus maniculatus that differ with regard to the number of sexual partners per individual to test the hypothesis that bacterial diversity should be greater in the promiscuous P. maniculatus relative to the monogamous P. californicus. As predicted, phylogenetically controlled and operational taxonomic unit-based indices of bacterial diversity indicated that diversity is greater in the promiscuous species. These results provide important new insights into the effects of mating system on bacterial diversity in free-living vertebrates, and may suggest a potential cost of promiscuity.

  19. NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism

    PubMed Central

    Kim, Youngchang; Cunningham, Mark A.; Mire, Joseph; Tesar, Christine; Sacchettini, James; Joachimiak, Andrzej

    2013-01-01

    The specter of a return to an era in which infectious disease looms as a significant threat to human health is not just hyperbole; there are serious concerns about the widespread overuse and misuse of antibiotics contributing to increased antibiotic resistance in pathogens. The recent discovery of a new enzyme, first identified in Klebsiella pneumoniae from a patient from New Delhi and denoted as NDM-1, represents an example of extreme promiscuity: It hydrolyzes and inactivates nearly all known β-lactam-based antibiotics with startling efficiency. NDM-1 can utilize different metal cofactors and seems to exploit an alternative mechanism based on the reaction conditions. Here we report the results of a combined experimental and theoretical study that examines the substrate, metal binding, and catalytic mechanism of the enzyme. We utilize structures obtained through X-ray crystallography, biochemical assays, and numerical simulation to construct a model of the enzyme catalytic pathway. The NDM-1 enzyme interacts with the substrate solely through zinc, or other metals, bound in the active site, explaining the observed lack of specificity against a broad range of β-lactam antibiotic agents. The zinc ions also serve to activate a water molecule that hydrolyzes the β-lactam ring through a proton shuttle.—Kim, Y., Cunningham, M. A.; Mire, J., Tesar, C., Sacchettini, J., Joachimiak, A. NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism. PMID:23363572

  20. Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis.

    PubMed

    Himes, Paul M; Allen, Scott E; Hwang, Sungwon; Bowers, Albert A

    2016-06-17

    Sactipeptides are peptide-derived natural products that are processed by remarkable, radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting sactionine thioether linkages give rise to the unique defined structures and concomitant biological activities of sactipeptides. An E. coli heterologous expression system, based on the biosynthesis of one such sactipeptide, subtilosin A, is described and this expression system is exploited to probe the promiscuity of the subtilosin A sactionine bond-forming enzyme, AlbA. These efforts allowed the facile expression and isolation of a small library of mutant sactipeptides based on the subtilosin A precursor peptide, demonstrating broad substrate promiscuity where none was previously known. Importantly, we show that the positions of the sactionine linkages can be moved, giving rise to new, unnatural sactipeptide structures. E. coli heterologous expression also allowed incorporation of unnatural amino acids into sactipeptides by means of amber-suppression technology, potentially opening up new chemistry and new applications for unnatural sactipeptides. PMID:27019323

  1. Tissue-specific expression of human arylsulfatase-C isozymes and steroid sulfatase.

    PubMed Central

    Munroe, D G; Chang, P L

    1987-01-01

    Steroid sulfatase (STS; E.C.3.1.6.2), which acts on 3-hydroxysteroid sulfates, and arylsulfatase-C (ARC; E.C.3.1.6.1), assayed with aromatic artificial substrates, are both membrane-bound, microsomal enzymes with alkaline pH optima. Although they copurify during preparation and their gene loci are mapped to the short arm of the human X chromosome where they appear to have escaped from X inactivation, it has not been settled whether STS and ARC are the same enzyme or not. Recent work from our laboratory has shown that ARC exists in two electrophoretically distinct forms in human fibroblasts. We now report that these two forms--the faster migrating (F) and more slowly migrating (S)--occur in human tissues. Each of 11 human tissue types from 10 subjects showed a consistent pattern of ARC isozymes. Thyroid, heart, spleen, skeletal muscle, and adrenal tissue mainly had the S form. In contrast, kidney, liver, and pancreas tissue had mainly the F form, while gonadal, lung, and intestinal tissue had both the S and the F forms. The question of escape of their gene locus from X-chromosome inactivation was examined by comparing the specific activities of ARC and STS in male-derived vis-à-vis female-derived tissues. The majority of the tissues did not show any significant difference in these activities between the sexes, the exceptions being heart muscle, gonadal, and kidney tissue. None showed the 1:2 ratio between male- and female-derived tissues expected of a locus that had escaped X inactivation. The question of identity between ARC and STS was examined by comparing the ratios of their activities in these tissue types: if the enzymes were identical, the ratios of their activities should have remained constant across the different tissue types. It was thus shown that ARC activity varied by as much as 100-fold, depending on the ARC isozymic pattern of the tissue. STS, measured as estrone sulfatase and dehydroepiandrosterone sulfatase, did not show similar variations. This

  2. Engineering stereocontrol into an aldolase-catalysed reaction.

    PubMed

    Lamble, Henry J; Danson, Michael J; Hough, David W; Bull, Steven D

    2005-01-01

    A novel thermostable aldolase has been developed for synthetic application, and substrate engineering has been used to induce stereocontrol into aldol reactions of this naturally-promiscuous enzyme. PMID:15614394

  3. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  4. Specificity, promiscuity, and the structure of complex information processing networks

    NASA Astrophysics Data System (ADS)

    Myers, Christopher

    2006-03-01

    Both the top-down designs of engineered systems and the bottom-up serendipities of biological evolution must negotiate tradeoffs between specificity and control: overly specific interactions between components can make systems brittle and unevolvable, while more generic interactions can require elaborate control in order to aggregate specificity from distributed pieces. Complex information processing systems reveal network organizations that navigate this landscape of constraints: regulatory and signaling networks in cells involve the coordination of molecular interactions that are surprisingly promiscuous, and object-oriented design in software systems emphasizes the polymorphic composition of objects of minimal necessary specificity [C.R. Myers, Phys Rev E 68, 046116 (2003)]. Models of information processing arising both in systems biology and engineered computation are explored to better understand how particular network organizations can coordinate the activity of promiscuous components to achieve robust and evolvable function.

  5. Unattractive, promiscuous and heavy drinkers: perceptions of women with tattoos.

    PubMed

    Swami, Viren; Furnham, Adrian

    2007-12-01

    This study examined social and physical perceptions of blonde and brunette women with different degrees of tattooing. Eighty-four female and 76 male undergraduates rated a series of 16 female line drawings that varied in 2 levels of hair colour and 8 levels of tattooing. Ratings were made for physical attractiveness and sexual promiscuity, as well as estimates of the number of alcohol units consumed on a typical night out. Results showed that tattooed women were rated as less physically attractive, more sexually promiscuous and heavier drinkers than untattooed women, with more negative ratings with increasing number of tattoos. There were also weak interactions between body art and hair colour, with blonde women in general rated more negatively than brunettes. Results are discussed in terms of stereotypes about women who have tattoos and the effects of such stereotypes on well-being. PMID:18089280

  6. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    PubMed Central

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-01-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer. PMID:24728108

  7. Crystal Structure and Promiscuous Partitioning of a Covalent Intermediate Common in the Pentein Superfamily

    PubMed Central

    Linsky, Thomas W.; Monzingo, Arthur F.; Stone, Everett M.; Robertus, Jon D.; Fast, Walter

    2008-01-01

    Summary Many enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here, we use a mutant (H162G) dimethylarginine dimethylaminohydrolase from Pseudomonas aeruginosa and an alternative substrate, S-methyl-L-thiocitrulline, to trap, crystallize and determine the 2.8 Å resolution structure of a stable covalent adduct which mimics this reaction intermediate. Observed interactions between the trapped adduct and active site residues along with comparison to a previously known product-bound structure provide insight into the normal catalytic mechanism. The plane of the trapped thiouronium intermediate is angled away from that seen in the product and substrate complexes, allowing for an altered angle of attack between the nucleophiles of the first and second half reactions. The stable covalent adduct is also capable of further reaction. Addition of exogenous imidazole can rescue the original hydrolytic activity. Notably, addition of other exogenous amines can instead yield substituted arginine products. These alternative products arise from partitioning of the trapped intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme scaffold provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities between different reaction pathways found in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this covalent intermediate may also help to illuminate the evolutionary history of these enzymes. PMID:18482699

  8. Mucopolysaccharidosis type IVA: Common double deletion in the N-Acetylgalactosamine-6-sulfatase gene (GALNS)

    SciTech Connect

    Hori, Toshinori; Tomatsu, Shunji; Fukuda, Seiji

    1995-04-10

    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster. 39 refs., 5 figs.

  9. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    NASA Astrophysics Data System (ADS)

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-06-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

  10. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound.

    PubMed

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q

    2015-01-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail. PMID:26105662

  11. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    PubMed Central

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-01-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail. PMID:26105662

  12. Promiscuous primates engage in same-sex genital interactions.

    PubMed

    MacFarlane, Geoff R; Vasey, Paul L

    2016-05-01

    Same-sex genital interactions (SSGIs) occur across the order primates, yet explaining their maintenance in evolutionary terms appears problematic; as such interactions seem to counteract reproductive goals. We hypothesised that in more promiscuous species, where sexual motivation, mating effort, and non-conceptive heterosexual behaviour are greater, SSGIs may also occur at greater frequencies without necessarily impeding reproduction. We found that the expression of both male and female SSGIs were greater in multimale systems than in unimale ones. Both male and female SSGIs were positively correlated with the degree of promiscuity (relative testes mass). As mating system confers biases in the sex ratio that may influence the expression of SSGIs, we controlled for availability of members of the same-sex. When employing this control, results were largely congruent. For males, SSGIs were expressed more frequently in multimale systems. For both sexes, SSGIs were expressed more frequently with greater relative testes mass. We suggest SSGIs in primates may be a neutral by-product of selection for increases in promiscuous sexual activity, and that in certain instances these interactions may be co-opted to facilitate adaptive social functions. PMID:26930251

  13. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  14. The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes.

    PubMed

    Landgrebe, Jobst; Dierks, Thomas; Schmidt, Bernhard; von Figura, Kurt

    2003-10-16

    Recently, the human C(alpha)-formylglycine (FGly)-generating enzyme (FGE), whose deficiency causes the autosomal-recessively transmitted lysosomal storage disease multiple sulfatase deficiency (MSD), has been identified. In sulfatases, FGE posttranslationally converts a cysteine residue to FGly, which is part of the catalytic site and is essential for sulfatase activity. FGE is encoded by the sulfatase modifying factor 1 (SUMF1) gene, which defines a new gene family comprising orthologs from prokaryotes to higher eukaryotes. The genomes of E. coli, S. cerevisiae and C. elegans lack SUMF1, indicating a phylogenetic gap and the existence of an alternative FGly-generating system. The genomes of vertebrates including mouse, man and pufferfish contain a sulfatase modifying factor 2 (SUMF2) gene encoding an FGE paralog of unknown function. SUMF2 evolved from a single exon SUMF1 gene as found in diptera prior to divergent intron acquisition. In several prokaryotic genomes, the SUMF1 gene is cotranscribed with genes encoding sulfatases which require FGly modification. The FGE protein contains a single domain that is made up of three highly conserved subdomains spaced by nonconserved sequences of variable lengths. The similarity among the eukaryotic FGE orthologs varies between 72% and 100% for the three subdomains and is highest for the C-terminal subdomain, which is a hotspot for mutations in MSD patients. PMID:14563551

  15. Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency

    PubMed Central

    Schlotawa, Lars; Radhakrishnan, Karthikeyan; Baumgartner, Matthias; Schmid, Regula; Schmidt, Bernhard; Dierks, Thomas; Gärtner, Jutta

    2013-01-01

    Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms. PMID:23321616

  16. Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Radhakrishnan, Karthikeyan; Baumgartner, Matthias; Schmid, Regula; Schmidt, Bernhard; Dierks, Thomas; Gärtner, Jutta

    2013-09-01

    Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms. PMID:23321616

  17. Transcriptional promiscuity of the human /alpha/-globin gene

    SciTech Connect

    Whitelaw, E.; Hogben, P.; Hanscombe, O.; Proudfoot, N.J.

    1989-01-01

    The human /alpha/-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. The authors have investigated whether there is a relationship between these two observations. First, they investigated the mouse /alpha/-globin gene since it does not lie in an HTF island. They have demonstrated that it was not transcriptionally promiscuous. Second, they studied the transcriptional activity of the human /alpha/-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human /alpha/-globin gene. They found that in a nonreplicating system, the human //a/-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since they only observed enhancer independence of the human /alpha/-globin gene in a high-copy-number replicating system, they suggest that competition for trans-acting factors could explain these results. Finally, the authors' experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human /alpha/-globin cap site or within the gene itself.

  18. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    PubMed

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. PMID:27208174

  19. Integrating mapping and sequencing around the human iduronate-2-sulfate sulfatase locus

    SciTech Connect

    Timms, K.; Lu, F.; Shen, Y.

    1994-09-01

    The logical progression of the human genome project is from mapping to sequencing. However, the criteria for accurate sequencing and mapping are different and consequently, sequencing can reveal unexpected or erroneous relationships between cosmid clones that appear overlapping by hybridization. We are sequencing a 1 Mb region of human Xq28 spanning the genes for fragile X (fraxA) and iduronate-2-sulfate sulfatase (IDS). To date, seven cosmids from this region have been completed and another five are currently being sequenced. One of the completed cosmids contains the complete IDS gene, while another cosmid contains 4 of the 9 IDS exons. The exon sequences in both cosmids are identical, but corresponding introns have proved to be highly variant. This raises the possibility of either a second IDS gene or unusual pseudogene. In addition, one of the cosmids contains a microsatellite marker which has been mapped 150 kb distant from the gene for IDS. This indicates that either two cosmids containing IDS exons are separated by at least 100 kb, or a rearrangement in one of the cosmids prior to library construction. To simplify the development of sequence-ready cosmids, we have developed a rapid method of cosmid walking to select additional clones that are minimally overlapping.

  20. Discovery of a sulfamate-based steroid sulfatase inhibitor with intrinsic selective estrogen receptor modulator properties.

    PubMed

    Ouellet, Charles; Maltais, René; Ouellet, Étienne; Barbeau, Xavier; Lagüe, Patrick; Poirier, Donald

    2016-08-25

    Steroid sulfatase (STS), the enzyme which converts inactive sulfated steroid precursors into active hormones, is a promising therapeutic target for the treatment of estrogen-sensitive breast cancer. We report herein the synthesis and in vitro study of dual-action STS inhibitors with selective estrogen-receptor modulator (SERM) effects. A library of tetrahydroisoquinoline-N-substituted derivatives (phenolic compounds) was synthesized by solid-phase chemistry and tested on estrogen-sensitive breast cancer T-47D cells. Three phenolic compounds devoid of estrogenic activity and toxicity emerged from this screening. Their sulfamate analogs were then synthesized, tested in STS-transfected HEK-293 cells, and found to be potent inhibitors of the enzyme (IC50 of 3.9, 8.9, and 16.6 nM). When tested in T-47D cells they showed no estrogenic activity and produced a moderate antiestrogenic activity. The compounds were further tested on osteoblast-like Saos-2 cells and found to significantly stimulate their proliferation as well as their alkaline phosphatase activity, thus suggesting a SERM activity. These results are supported by molecular docking experiments. PMID:27155470

  1. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J; Pimentel, Luisa; Barrera, Luis A

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  2. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  3. Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma

    PubMed Central

    Phillips, Joanna J.; Huillard, Emmanuelle; Robinson, Aaron E.; Ward, Anna; Lum, David H.; Polley, Mei-Yin; Rosen, Steven D.; Rowitch, David H.; Werb, Zena

    2012-01-01

    Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2–/– tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor–mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM. PMID:22293178

  4. Maintenance of Sperm Variation in a Highly Promiscuous Wild Bird

    PubMed Central

    Calhim, Sara; Double, Michael C.; Margraf, Nicolas; Birkhead, Tim R.; Cockburn, Andrew

    2011-01-01

    Postcopulatory sexual selection is an important force in the evolution of reproductive traits, including sperm morphology. In birds, sperm morphology is known to be highly heritable and largely condition-independent. Theory predicts, and recent comparative work corroborates, that strong selection in such traits reduces intraspecific phenotypic variation. Here we show that some variation can be maintained despite extreme promiscuity, as a result of opposing, copulation-role-specific selection forces. After controlling for known correlates of siring success in the superb fairy-wren (Malurus cyaneus), we found that (a) lifetime extra-pair paternity success was associated with sperm with a shorter flagellum and relatively large head, and (b) males whose sperm had a longer flagellum and a relatively smaller head achieved higher within-pair paternity. In this species extrapair copulations occur in the same morning, but preceding, pair copulations during a female's fertile period, suggesting that shorter and relatively larger-headed sperm are most successful in securing storage (defense), whereas the opposite phenotype might be better at outcompeting stored sperm (offense). Furthermore, since cuckolding ability is a major contributor to differential male reproductive output, stronger selection on defense sperm competition traits might explain the short sperm of malurids relative to other promiscuous passerines. PMID:22194918

  5. Tailoring Agility: Promiscuous Pair Story Authoring and Value Calculation

    NASA Astrophysics Data System (ADS)

    Tendon, Steve

    This chapter describes how a multi-national software organization created a business plan involving business units from eight countries that followed an agile way, after two previously failed attempts with traditional approaches. The case is told by the consultant who initiated implementation of agility into requirements gathering, estimation and planning processes in an international setting. The agile approach was inspired by XP, but then tailored to meet the peculiar requirements. Two innovations were critical. The first innovation was promiscuous pair story authoring, where user stories were written by two people (similarly to pair programming), and the pairing changed very often (as frequently as every 15-20 minutes) to achieve promiscuity and cater for diverse point of views. The second innovation was an economic value evaluation (and not the cost) which was attributed to stories. Continuous recalculation of the financial value of the stories allowed to assess the projects financial return. In this case implementation of agility in the international context allowed the involved team members to reach consensus and unanimity of decisions, vision and purpose.

  6. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    PubMed

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  7. Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

    PubMed

    Devine, Shane M; Mulcair, Mark D; Debono, Cael O; Leung, Eleanor W W; Nissink, J Willem M; Lim, San Sui; Chandrashekaran, Indu R; Vazirani, Mansha; Mohanty, Biswaranjan; Simpson, Jamie S; Baell, Jonathan B; Scammells, Peter J; Norton, Raymond S; Scanlon, Martin J

    2015-02-12

    We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries. PMID:25559643

  8. Expression in CHO Cells and Pharmacokinetics and Brain Uptake in the Rhesus Monkey of an IgG-Iduronate-2-Sulfatase Fusion Protein

    PubMed Central

    Lu, Jeff Zhiqiang; Boado, Ruben J.; Hui, Eric K.-W.; Zhou, Qing-Hui; Pardridge, William M.

    2011-01-01

    Sulfatases are potential therapeutic biopharmaceuticals, as mutations in sulfatase genes leads to inherited disease. Mucopolysaccharidosis (MPS) Type II is caused by mutations in the lysosomal enzyme, iduronate 2-sulfatase (IDS). MPS-II affects the brain and enzyme replacement therapy is ineffective for the brain, because IDS does not cross the blood-brain barrier (BBB). To deliver IDS across the human BBB, the sulfatase has been re-engineered as an IgG-sulfatase fusion protein with a genetically engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb part of the HIRMAb-IDS fusion protein acts as a molecular Trojan horse to ferry the fused IDS across the BBB. Chinese hamster ovary (CHO) cell were stably transfected to produce the HIRMAb-IDS fusion protein. The fusion protein was triaged to the lysosomal compartment of MPS-II fibroblasts based on confocal microscopy, and 300 ng/mL medium concentrations normalized IDS enzyme activity in the cells. The HIRMAb-IDS fusion protein was tritiated and injected intravenously into the adult Rhesus monkey at a low dose of 0.1 mg/kg. The IDS enzyme activity in plasma was elevated 10-fold above the endogenous level, and therapeutic plasma concentrations were generated in vivo. The uptake of the HIRMAb-IDS fusion protein in the brain was sufficiently high to produce therapeutic concentrations of IDS in the brain following IV administration of the fusion protein. PMID:21351076

  9. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles.

    PubMed

    Fornili, Arianna; Pandini, Alessandro; Lu, Hui-Chun; Fraternali, Franca

    2013-11-12

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

  10. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

    PubMed Central

    2013-01-01

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Catalytic Promiscuity of the Radical S-adenosyl-L-methionine Enzyme NosL

    PubMed Central

    Ding, Wei; Ji, Xinjian; Li, Yongzhen; Zhang, Qi

    2016-01-01

    Catalytic promiscuity plays a key role in enzyme evolution and the acquisition of novel biological functions. Because of the high reactivity of radical species, in our view enzymes involving radical-mediated mechanisms could intrinsically be more prone to catalytic promiscuity. This mini-review summarizes the recent advances in the study of NosL, a radical S-adenosyl-L-methionine (SAM)-dependent L-tryptophan (L-Trp) lyase. We demonstrate here the interesting chemistry and remarkable catalytic promiscuity of NosL, and attempt to highlight the high evolvability of radical SAM enzymes and the potential to engineer these enzymes for novel and improved activities. PMID:27446906

  14. Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume.

    PubMed

    Billich, Andreas; Bilban, Melitta; Meisner, Nicole-Claudia; Nussbaumer, Peter; Neubauer, Andreas; Jäger, Stefan; Auer, Manfred

    2004-02-01

    Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening. PMID:15090207

  15. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species

    PubMed Central

    Dasmahapatra, Kanchon K; Walters, James R.; Briscoe, Adriana D.; Davey, John W.; Whibley, Annabel; Nadeau, Nicola J.; Zimin, Aleksey V.; Hughes, Daniel S. T.; Ferguson, Laura C.; Martin, Simon H.; Salazar, Camilo; Lewis, James J.; Adler, Sebastian; Ahn, Seung-Joon; Baker, Dean A.; Baxter, Simon W.; Chamberlain, Nicola L.; Chauhan, Ritika; Counterman, Brian A.; Dalmay, Tamas; Gilbert, Lawrence E.; Gordon, Karl; Heckel, David G.; Hines, Heather M.; Hoff, Katharina J.; Holland, Peter W.H.; Jacquin-Joly, Emmanuelle; Jiggins, Francis M.; Jones, Robert T.; Kapan, Durrell D.; Kersey, Paul; Lamas, Gerardo; Lawson, Daniel; Mapleson, Daniel; Maroja, Luana S.; Martin, Arnaud; Moxon, Simon; Palmer, William J.; Papa, Riccardo; Papanicolaou, Alexie; Pauchet, Yannick; Ray, David A.; Rosser, Neil; Salzberg, Steven L.; Supple, Megan A.; Surridge, Alison; Tenger-Trolander, Ayse; Vogel, Heiko; Wilkinson, Paul A.; Wilson, Derek; Yorke, James A.; Yuan, Furong; Balmuth, Alexi L.; Eland, Cathlene; Gharbi, Karim; Thomson, Marian; Gibbs, Richard A.; Han, Yi; Jayaseelan, Joy C.; Kovar, Christie; Mathew, Tittu; Muzny, Donna M.; Ongeri, Fiona; Pu, Ling-Ling; Qu, Jiaxin; Thornton, Rebecca L.; Worley, Kim C.; Wu, Yuan-Qing; Linares, Mauricio; Blaxter, Mark L.; Constant, Richard H. ffrench; Joron, Mathieu; Kronforst, Marcus R.; Mullen, Sean P.; Reed, Robert D.; Scherer, Steven E.; Richards, Stephen; Mallet, James; McMillan, W. Owen; Jiggins, Chris D.

    2012-01-01

    The evolutionary importance of hybridization and introgression has long been debated1. We used genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation2-5 . We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,657 predicted genes for Heliconius, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organisation has remained broadly conserved since the Cretaceous, when butterflies split from the silkmoth lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, H. melpomene, H. timareta, and H. elevatus, especially at two genomic regions that control mimicry pattern. Closely related Heliconius species clearly exchange protective colour pattern genes promiscuously, implying a major role for hybridization in adaptive radiation. PMID:22722851

  16. Alcohol and adult hippocampal neurogenesis: Promiscuous drug, wanton effects

    PubMed Central

    Geil, Chelsea R.; Hayes, Dayna M.; McClain, Justin A.; Liput, Daniel J.; Marshall, S. Alex; Chen, Kevin Y.; Nixon, Kimberly

    2014-01-01

    Adult neurogenesis is now widely accepted as an important contributor to hippocampal integrity and function but also dysfunction when adult neurogenesis is affected in neuropsychiatric diseases such as alcohol use disorders. Excessive alcohol consumption, the defining characteristic of alcohol use disorders, results in a variety of cognitive and behavioral impairments related wholly or in part to hippocampal structure and function. Recent preclinical work has shown that adult neurogenesis may be one route by which alcohol produces hippocampal neuropathology. Alcohol is a pharmacologically promiscuous drug capable of interfering with adult neurogenesis through multiple mechanisms. This review will discuss the primary mechanisms underlying alcohol-induced changes in adult hippocampal neurogenesis including alcohol's effects on neurotransmitters, CREB and its downstream effectors, and the neurogenic niche. PMID:24842804

  17. Circadian transcriptome analysis in human fibroblasts from Hunter syndrome and impact of iduronate-2-sulfatase treatment

    PubMed Central

    2013-01-01

    Background Hunter syndrome (HS) is a lysosomal storage disease caused by iduronate-2-sulfatase (IDS) deficiency and loss of ability to break down and recycle the glycosaminoglycans, heparan and dermatan sulfate, leading to impairment of cellular processes and cell death. Cell activities and functioning of intracellular organelles are controlled by the clock genes (CGs), driving the rhythmic expression of clock controlled genes (CCGs). We aimed to evaluate the expression of CGs and downstream CCGs in HS, before and after enzyme replacement treatment with IDS. Methods The expression levels of CGs and CCGs were evaluated by a whole transcriptome analysis through Next Generation Sequencing in normal primary human fibroblasts and fibroblasts of patients affected by HS before and 24 h/144 h after IDS treatment. The time related expression of CGs after synchronization by serum shock was also evaluated by qRT-PCR before and after 24 hours of IDS treatment. Results In HS fibroblasts we found altered expression of several CGs and CCGs, with dynamic changes 24 h and 144 h after IDS treatment. A semantic hypergraph-based analysis highlighted five gene clusters significantly associated to important biological processes or pathways, and five genes, AHR, HIF1A, CRY1, ITGA5 and EIF2B3, proven to be central players in these pathways. After synchronization by serum shock and 24 h treatment with IDS the expression of ARNTL2 at 10 h (p = 0.036), PER1 at 4 h (p = 0.019), PER2 at 10 h (p = 0.041) and 16 h (p = 0.043) changed in HS fibroblasts. Conclusion CG and CCG expression is altered in HS fibroblasts and IDS treatment determines dynamic modifications, suggesting a direct involvement of the CG machinery in the physiopathology of cellular derangements that characterize HS. PMID:24083598

  18. The Role of Steroid Sulfatase as a Prognostic Factor in Patients with Endometrial Cancer

    PubMed Central

    Lee, Won Moo; Jang, Ki-Seok; Koh, A Ra

    2016-01-01

    Purpose The aim of the study was to determine steroid sulfatase (STS) expression in endometrial cancer patients and its correlation with disease prognosis. Materials and Methods We conducted a retrospective study in 59 patients who underwent surgery with histologically confirmed endometrial cancer from January 2000 to December 2011 at Hanyang University Hospital. Immuno-histochemical staining of STS was performed using rabbit polyclonal anti-STS antibody. Results Sixteen of the 59 patients (27.1%) were positive for STS expression. Disease free survival (DFS) was 129.83±8.67 [95% confidence interval (CI): 112.84–146.82] months in the STS positive group (group A) and 111.06±7.17 (95% CI: 97.01–125.10) months in the STS negative group (group B) (p=0.92). Overall survival (OS) was 129.01±9.38 (95% CI: 110.63–147.38) months and 111.16±7.10 (95% CI: 97.24–125.07) months for the groups A and B, respectively (p=0.45). Univariate analysis revealed that FIGO stage and adjuvant therapy are significantly associated with DFS and OS. However, in multivariate analysis, FIGO stage and adjuvant therapy did not show any statistical significance with DFS and OS. STS was also not significantly associated with DFS and OS in univariate and multivariate analysis. Conclusion STS expression was not significantly associated with DFS and OS, despite positive STS expression in 27% of endometrial cancer patients. Therefore, the role of STS as a prognostic factor in patients with endometrial cancer remains unclear and requires further research. PMID:26996578

  19. Catch bond interaction between cell-surface sulfatase Sulf1 and glycosaminoglycans.

    PubMed

    Harder, Alexander; Möller, Ann-Kristin; Milz, Fabian; Neuhaus, Phillipp; Walhorn, Volker; Dierks, Thomas; Anselmetti, Dario

    2015-04-01

    In biological adhesion, the biophysical mechanism of specific biomolecular interaction can be divided in slip and catch bonds, respectively. Conceptually, slip bonds exhibit a reduced bond lifetime under increased external force and catch bonds, in contrast, exhibit an increased lifetime (for a certain force interval). Since 2003, a handful of biological systems have been identified to display catch bond properties. Upon investigating the specific interaction between the unique hydrophilic domain (HD) of the human cell-surface sulfatase Sulf1 against its physiological glycosaminoglycan (GAG) target heparan sulfate (HS) by single molecule force spectroscopy (SMFS), we found clear evidence of catch bond behavior in this system. The HD, ∼320 amino acids long with dominant positive charge, and its interaction with sulfated GAG-polymers were quantitatively investigated using atomic force microscopy (AFM) based force clamp spectroscopy (FCS) and dynamic force spectroscopy (DFS). In FCS experiments, we found that the catch bond character of HD against GAGs could be attributed to the GAG 6-O-sulfation site whereas only slip bond interaction can be observed in a GAG system where this site is explicitly lacking. We interpreted the binding data within the theoretical framework of a two state two path model, where two slip bonds are coupled forming a double-well interaction potential with an energy difference of ΔE ≈ 9 kBT and a compliance length of Δx ≈ 3.2 nm. Additional DFS experiments support this assumption and allow identification of these two coupled slip-bond states that behave consistently within the Kramers-Bell-Evans model of force-mediated dissociation. PMID:25863062

  20. The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

    PubMed

    Dalla Valle, Luisa; Toffolo, Vania; Nardi, Alessia; Fiore, Cristina; Armanini, Decio; Belvedere, Paola; Colombo, Lorenzo

    2007-10-01

    We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration. PMID:17601726

  1. Analyzing compound activity records and promiscuity degrees in light of publication statistics

    PubMed Central

    Hu, Ye; Bajorath, Jürgen

    2016-01-01

    For the generation of contemporary databases of bioactive compounds, activity information is usually extracted from the scientific literature. However, when activity data are analyzed, source publications are typically no longer taken into consideration. Therefore, compound activity data selected from ChEMBL were traced back to thousands of original publications, activity records including compound, assay, and target information were systematically generated, and their distributions across the literature were determined. In addition, publications were categorized on the basis of activity records. Furthermore, compound promiscuity, defined as the ability of small molecules to specifically interact with multiple target proteins, was analyzed in light of publication statistics, thus adding another layer of information to promiscuity assessment. It was shown that the degree of compound promiscuity was not influenced by increasing numbers of source publications. Rather, most non-promiscuous as well as promiscuous compounds, regardless of their degree of promiscuity, originated from single publications, which emerged as a characteristic feature of the medicinal chemistry literature. PMID:27347396

  2. Biological messiness vs. biological genius: Mechanistic aspects and roles of protein promiscuity.

    PubMed

    Atkins, William M

    2015-07-01

    In contrast to the traditional biological paradigms focused on 'specificity', recent research and theoretical efforts have focused on functional 'promiscuity' exhibited by proteins and enzymes in many biological settings, including enzymatic detoxication, steroid biochemistry, signal transduction and immune responses. In addition, divergent evolutionary processes are apparently facilitated by random mutations that yield promiscuous enzyme intermediates. The intermediates, in turn, provide opportunities for further evolution to optimize new functions from existing protein scaffolds. In some cases, promiscuity may simply represent the inherent plasticity of proteins resulting from their polymeric nature with distributed conformational ensembles. Enzymes or proteins that bind or metabolize noncognate substrates create 'messiness' or noise in the systems they contribute to. With our increasing awareness of the frequency of these promiscuous behaviors it becomes interesting and important to understand the molecular bases for promiscuous behavior and to distinguish between evolutionarily selected promiscuity and evolutionarily tolerated messiness. This review provides an overview of current understanding of these aspects of protein biochemistry and enzymology. PMID:25218442

  3. A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis

    PubMed Central

    Abitbol, Marie; Thibaud, Jean-Laurent; Olby, Natasha J.; Hitte, Christophe; Puech, Jean-Philippe; Maurer, Marie; Pilot-Storck, Fanny; Hédan, Benoit; Dréano, Stéphane; Brahimi, Sandra; Delattre, Delphine; André, Catherine; Gray, Françoise; Delisle, Françoise; Caillaud, Catherine; Bernex, Florence; Panthier, Jean-Jacques; Aubin-Houzelstein, Geneviève; Tiret, Laurent

    2010-01-01

    Neuronal ceroid lipofuscinoses (NCLs) represent the most common group of inherited progressive encephalopathies in children. They are characterized by progressive loss of vision, mental and motor deterioration, epileptic seizures, and premature death. Rare adult forms of NCL with late onset are known as Kufs’ disease. Loci underlying these adult forms remain unknown due to the small number of patients and genetic heterogeneity. Here we confirm that a late-onset form of NCL recessively segregates in US and French pedigrees of American Staffordshire Terrier (AST) dogs. Through combined association, linkage, and haplotype analyses, we mapped the disease locus to a single region of canine chromosome 9. We eventually identified a worldwide breed-specific variant in exon 2 of the Arylsulfatase G (ARSG) gene, which causes a p.R99H substitution in the vicinity of the catalytic domain of the enzyme. In transfected cells or leukocytes from affected dogs, the missense change leads to a 75% decrease in sulfatase activity, providing a functional confirmation that the variant might be the NCL-causing mutation. Our results uncover a protein involved in neuronal homeostasis, identify a family of candidate genes to be screened in patients with Kufs' disease, and suggest that a deficiency in sulfatase is part of the NCL pathogenesis. PMID:20679209

  4. Determination of the molecular defect of caprine N-acetylglucosamine 6-sulfatase deficiency

    SciTech Connect

    Leipprandt, J.R.; Jones, M.Z.; Cavanagh, K.T.

    1994-09-01

    Caprine N-acetylglucosamine 6-sulfatase (G6S) deficiency is the only animal analog of Sanfilippo syndrome (type D). The goat with this mucopolysaccharidousis disorder (MPS III D) demonstrated delayed motor development and growth retardation but reached sexual maturity before dying suddenly at 19 mo. Histochemical and biochemical analysis of the liver showed glycosaminoglycan storage and there was GM{sub 3} ganglioside accumulation in the brain. Towards further development of this animal model for treatment strategies, we have cloned the caprine G6S gene, determined the nature of the gene defect in caprine MPS III D and compared the goat sequence to the human sequence. The human and caprine sequences show an overall sequence similarity of about 90% in the coding region. The 5{prime}-coding region is very GC-rich in both the human and caprine G6S. One striking difference between the human and caprine genes is the presence of a GCC repeat in the goat resulting in insertion of 6 prolines and a leucine in the signal peptide. This proline-rich stretch was confirmed by amplifying and sequencing the same cDNA segment from other goats. Additionally, this region was examined in bovine cDNA and found to contain 4 prolines and 2 leucines. The mRNA for G6S consists of two species of approximately 4.0 and 4.2 kb with a coding region of 1.6 kb. For mutation analysis a series of primers was designed to cover the entire G6S coding region. Amplicons from RT-PCR on normal and affected goat total RNA were produced and sequenced. A single base substitution, T for C, was found in the 5{prime} region of the coding sequence of the affected animals that creates a stop codon. This mutation introduces an Alu I restriction site. PCR primers designed to amplify a short segment of genomic DNA encompassing the mutation have been used to identify putative carriers and develop a caprine Sanfilippo III D carrier colony.

  5. Stratum corneum lipids in disorders of cornification. Steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis.

    PubMed Central

    Elias, P M; Williams, M L; Maloney, M E; Bonifas, J A; Brown, B E; Grayson, S; Epstein, E H

    1984-01-01

    The pathological scaling in recessive x-linked ichthyosis is associated with accumulation of abnormal quantities of cholesterol sulfate in stratum corneum (J. Clin. Invest. 68:1404-1410, 1981). To determine whether or not cholesterol sulfate accumulates in recessive x-linked ichthyosis as a direct result of the missing enzyme, steroid sulfatase, we quantitated both steroid sulfatase and its substrate, we quantitated both steroid sulfatase and its substrate, cholesterol sulfate, in different epidermal strata, as well as within stratum corneum subcellular fractions obtained from normal human and neonatal mouse epidermis and from patients with recessive x-linked ichthyosis. In normal human and mouse epidermis, steroid sulfatase activity peaked in the stratum granulosum and stratum corneum, and negligible activity was detectable in lower epidermal layers. In contrast, in recessive x-linked ichthyosis epidermis, enzyme levels were virtually undetectable at all levels. In normal human stratum corneum, up to 10 times more steroid sulfatase activity was present in purified peripheral membrane preparations than in the whole tissue. Whereas in normal human epidermis cholesterol sulfate levels were lowest in the basal/spinous layer, and highest in the stratum granulosum, in recessive x-linked ichthyosis the levels were only slightly higher in the lower epidermis, but continued to climb in the stratum corneum. In both normal and in recessive x-linked ichthyosis stratum corneum, cholesterol sulfate appeared primarily within membrane domains, paralleling the pattern of steroid sulfatase localization. Finally, the role of excess cholesterol sulfate in the pathogenesis of recessive x-linked ichthyosis was directly tested by topical applications of this substance, which produced visible scaling in hairless mice in parallel to an increased cholesterol sulfate content of the stratum corneum. These results demonstrate an intimate relationship between steroid sulfatase and cholesterol

  6. Sulfatases and a Radical S-Adenosyl-l-methionine (AdoMet) Enzyme Are Key for Mucosal Foraging and Fitness of the Prominent Human Gut Symbiont, Bacteroides thetaiotaomicron*

    PubMed Central

    Benjdia, Alhosna; Martens, Eric C.; Gordon, Jeffrey I.; Berteau, Olivier

    2011-01-01

    The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Nevertheless, the molecular basis of the interactions between commensal bacteria and their host remained to be investigated. Bacteria colonizing the mucosal layer that overlies the gut epithelium are exposed to highly sulfated glycans (i.e. mucin and glycosaminoglycans). These polymers can serve as potential nutrient sources, but their high sulfate content usually prevents their degradation. Commensal bacteria such as Bacteroides thetaiotaomicron possess more predicted sulfatase genes than in the human genome, the physiological functions of which are largely unknown. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the radical AdoMet enzyme anaerobic sulfatase-maturating enzyme (anSME). In the present study, we have tested the role of this pathway in Bacteroides thetaiotaomicron which, in addition to 28 predicted sulfatases, possesses a single predicted anSME. In vitro studies revealed that deletion of the gene encoding its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of formerly germ-free mice with both isogenic strains (i.e. wild-type or ΔanSME), or invasion experiments involving introduction of one followed by the other strain established that anSME activity and the sulfatases activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans

  7. Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

    PubMed

    Dierks, Thomas; Schlotawa, Lars; Frese, Marc-André; Radhakrishnan, Karthikeyan; von Figura, Kurt; Schmidt, Bernhard

    2009-04-01

    Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been

  8. Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Steinfeld, Robert; von Figura, Kurt; Dierks, Thomas; Gärtner, Jutta

    2008-01-01

    Multiple Sulfatase Deficiency (MSD) is a rare inborn autosomal-recessive disorder, which mainly combines clinical features of metachromatic leukodystrophy, mucopolysaccharidosis and X-linked ichthyosis. The clinical course ranges from neonatal severe to mild juvenile cases. MSD is caused by mutations in the SUMF1 gene encoding the formylglycine-generating enzyme (FGE). FGE posttranslationally activates sulfatases by generating formylglycine in their catalytic sites. We analyzed the functional consequences of missense mutations p.A177P, p.W179S, p.A279V and p.R349W with regard to FGE's subcellular localization, enzymatic activity, protein stability, intracellular retention and resulting sulfatase activities. All four mutations did not affect localization of FGE in the endoplasmic reticulum of MSD fibroblasts. However, they decreased its specific enzymatic activity to less than 1% (p.A177P and p.R349W), 3% (p.W179S) or 23% (p.A279V). Protein stability was severely decreased for p.A279V and p.R349W, and almost comparable to wild type for p.A177P and p.W179S. The patient with the mildest clinical phenotype carries the mutation p.A279V leading to decreased FGE protein stability, but high residual enzymatic activity and only slightly reduced sulfatase activities. In contrast, the most severely affected patient carries the mutation p.R349W leading to drastically decreased protein stability, very low residual enzymatic activity and considerably reduced sulfatase activities. Our functional studies provide novel insight into the molecular defect underlying MSD and reveal that both residual enzyme activity and protein stability of FGE contribute to the clinical phenotype. The application of improved functional assays to determine these two molecular parameters of FGE mutants may enable the prediction of the clinical outcome in the future. PMID:18157819

  9. Legume-rhizobia signal exchange: promiscuity and environmental effects

    PubMed Central

    Lira, Mario A.; Nascimento, Luciana R. S.; Fracetto, Giselle G. M.

    2015-01-01

    Although signal exchange between legumes and their rhizobia is among the best-known examples of this biological process, most of the more characterized data comes from just a few legume species and environmental stresses. Although a relative wealth of information is available for some model legumes and some of the major pulses such as soybean, little is known about tropical legumes. This relative disparity in current knowledge is also apparent in the research on the effects of environmental stress on signal exchange; cool-climate stresses, such as low-soil temperature, comprise a relatively large body of research, whereas high-temperature stresses and drought are not nearly as well understood. Both tropical legumes and their environmental stress-induced effects are increasingly important due to global population growth (the demand for protein), climate change (increasing temperatures and more extreme climate behavior), and urbanization (and thus heavy metals). This knowledge gap for both legumes and their environmental stresses is compounded because whereas most temperate legume-rhizobia symbioses are relatively specific and cultivated under relatively stable environments, the converse is true for tropical legumes, which tend to be promiscuous, and grow in highly variable conditions. This review will clarify some of this missing information and highlight fields in which further research would benefit our current knowledge. PMID:26441880

  10. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  11. The energetic cost of mating in a promiscuous cephalopod.

    PubMed

    Franklin, Amanda Michelle; Squires, Zoe Elizabeth; Stuart-Fox, Devi

    2012-10-23

    Costs that individuals incur through mating can play an important role in understanding the evolution of life histories and senescence, particularly in promiscuous species. Copulation costs, ranging from energy expenditure to reduced longevity, are widely studied in insects but have received substantially less attention in other taxa. One cost of mating, the energetic cost, is poorly studied across all taxa despite its potential importance for the many species where copulation is physically demanding and/or frequent. Here, we investigated the energetic cost of mating in both male and female dumpling squid (Euprymna tasmanica). In this species, copulation can last up to 3 h and requires that the male physically restrains the female. We report that the act of copulation halves the swimming endurance of both sexes, and that they take up to 30 min to recover. Such a reduction in post-copulatory performance may have important implications for predator avoidance, foraging ability and energy allocation. Therefore, quantifying this cost is essential to understand the evolution of reproductive strategies and behaviours such as female receptivity and male and female mating frequency. PMID:22809722

  12. MTH1 Substrate Recognition--An Example of Specific Promiscuity.

    PubMed

    Nissink, J Willem M; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  13. The mammalian mineralocorticoid receptor: tying down a promiscuous receptor.

    PubMed

    Gomez-Sanchez, Elise P

    2010-01-01

    The mineralocorticoid receptor (MR) has been called a promiscuous receptor because its intrinsic affinity for aldosterone, cortisol and corticosterone is similar. Since glucocorticoids circulate in concentrations 100- to 1000-fold those of aldosterone, stoichiometry dictates that MR should be activated by glucocorticoids, not aldosterone, yet MRs are expressed in many tissues and regulate diverse functions, many of them under the regulation of the renin-angiotensin-aldosterone system. A relatively small number of brain MRs are aldosterone selective and modulate blood pressure. Evidence for possible mechanisms conferring ligand specificity in the context of mineralocorticoid-induced hypertension and the brain are discussed. These include factors (or mechanisms) intrinsic to the receptor, such as alternative splice variants and translation start sites, and extrinsic to the MR, including differential access through the blood-brain barrier, differential recruitment of co-regulators and scaffolding proteins, 11beta-steroid dehydrogenase activity, synthesis of potent acylated aldosterone derivatives and the synthesis of relevant amounts of aldosterone in areas of the brain that modulate blood pressure. PMID:19648477

  14. Legume-rhizobia signal exchange: promiscuity and environmental effects.

    PubMed

    Lira, Mario A; Nascimento, Luciana R S; Fracetto, Giselle G M

    2015-01-01

    Although signal exchange between legumes and their rhizobia is among the best-known examples of this biological process, most of the more characterized data comes from just a few legume species and environmental stresses. Although a relative wealth of information is available for some model legumes and some of the major pulses such as soybean, little is known about tropical legumes. This relative disparity in current knowledge is also apparent in the research on the effects of environmental stress on signal exchange; cool-climate stresses, such as low-soil temperature, comprise a relatively large body of research, whereas high-temperature stresses and drought are not nearly as well understood. Both tropical legumes and their environmental stress-induced effects are increasingly important due to global population growth (the demand for protein), climate change (increasing temperatures and more extreme climate behavior), and urbanization (and thus heavy metals). This knowledge gap for both legumes and their environmental stresses is compounded because whereas most temperate legume-rhizobia symbioses are relatively specific and cultivated under relatively stable environments, the converse is true for tropical legumes, which tend to be promiscuous, and grow in highly variable conditions. This review will clarify some of this missing information and highlight fields in which further research would benefit our current knowledge. PMID:26441880

  15. Phytochemicals perturb membranes and promiscuously alter protein function.

    PubMed

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  16. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species.

    PubMed

    2012-07-01

    The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation. PMID:22722851

  17. A promiscuous DNA packaging machine from bacteriophage T4.

    PubMed

    Zhang, Zhihong; Kottadiel, Vishal I; Vafabakhsh, Reza; Dai, Li; Chemla, Yann R; Ha, Taekjip; Rao, Venigalla B

    2011-01-01

    Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles. PMID:21358801

  18. Beyond promiscuity: mate-choice commitments in social breeding

    PubMed Central

    Boomsma, Jacobus J.

    2013-01-01

    Obligate eusociality with distinct caste phenotypes has evolved from strictly monogamous sub-social ancestors in ants, some bees, some wasps and some termites. This implies that no lineage reached the most advanced form of social breeding, unless helpers at the nest gained indirect fitness values via siblings that were identical to direct fitness via offspring. The complete lack of re-mating promiscuity equalizes sex-specific variances in reproductive success. Later, evolutionary developments towards multiple queen-mating retained lifetime commitment between sexual partners, but reduced male variance in reproductive success relative to female's, similar to the most advanced vertebrate cooperative breeders. Here, I (i) discuss some of the unique and highly peculiar mating system adaptations of eusocial insects; (ii) address ambiguities that remained after earlier reviews and extend the monogamy logic to the evolution of soldier castes; (iii) evaluate the evidence for indirect fitness benefits driving the dynamics of (in)vertebrate cooperative breeding, while emphasizing the fundamental differences between obligate eusociality and cooperative breeding; (iv) infer that lifetime commitment is a major driver towards higher levels of organization in bodies, colonies and mutualisms. I argue that evolutionary informative definitions of social systems that separate direct and indirect fitness benefits facilitate transparency when testing inclusive fitness theory. PMID:23339241

  19. MTH1 Substrate Recognition—An Example of Specific Promiscuity

    PubMed Central

    Nissink, J. Willem M.; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J.

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  20. Promiscuous mating in the harem-roosting fruit bat, Cynopterus sphinx.

    PubMed

    Garg, Kritika M; Chattopadhyay, Balaji; Doss D, Paramanatha Swami; A K, Vinoth Kumar; Kandula, Sripathi; Ramakrishnan, Uma

    2012-08-01

    Observations on mating behaviours and strategies guide our understanding of mating systems and variance in reproductive success. However, the presence of cryptic strategies often results in situations where social mating system is not reflective of genetic mating system. We present such a study of the genetic mating system of a harem-forming bat Cynopterus sphinx where harems may not be true indicators of male reproductive success. This temporal study using data from six seasons on paternity reveals that social harem assemblages do not play a role in the mating system, and variance in male reproductive success is lower than expected assuming polygynous mating. Further, simulations reveal that the genetic mating system is statistically indistinguishable from promiscuity. Our results are in contrast to an earlier study that demonstrated high variance in male reproductive success. Although an outcome of behavioural mating patterns, standardized variance in male reproductive success (I(m)) affects the opportunity for sexual selection. To gain a better understanding of the evolutionary implications of promiscuity for mammals in general, we compared our estimates of I(m) and total opportunity for sexual selection (I(m) /I(f), where I(f) is standardized variance in female reproductive success) with those of other known promiscuous species. We observed a broad range of I(m) /I(f) values across known promiscuous species, indicating our poor understanding of the evolutionary implications of promiscuous mating. PMID:22725709

  1. The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes.

    PubMed

    Martínez-Núñez, Mario Alberto; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

    2015-09-01

    The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free-living, extremophiles, pathogens, and intracellular. From these analyses we found that free-living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free-living and extremophiles species. PMID:26109005

  2. Morquio A syndrome: Cloning, sequence, and structure of the human N-acetylgalactosamine 6-sulfatase (GALNS) gene

    SciTech Connect

    Morris, C.P.; Guo, Xiao-Hui; Apostolou, S.

    1994-08-01

    Deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulfatase (GALNS;EC 3.1.6.4), results in the storage of the glycosaminoglycans, keratan sulfate and chrondroitin 6-sulfate, which leads to the lysosomal storage disorder Morquio A syndrome. Four overlapping genomic clones derived from a chromosome 16-specific gridded cosmid library containing the entire GALNS gene were isolated. The structure of the gene and the sequence of the exon/intron boundaries and the 5{prime} promoter region were determined. The GALNS gene is split into 14 exons spanning approximately 40 kb. The potential promoter for GALNS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. The GALNS gene contains an Alu repeat in intron 5 and a VNTR-like sequence in intron 6. 12 refs., 3 figs., 1 tab.

  3. Structural Basis for Substrate Promiscuity of dCK

    SciTech Connect

    Sabini, Elisabetti; Hazra, Saugata; Ort, Stephen; Konrad, Manfred; Lavie, Arnon

    2008-06-06

    Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.

  4. Filtering promiscuous compounds in early drug discovery: is it a good idea?

    PubMed

    Senger, Mario R; Fraga, Carlos A M; Dantas, Rafael F; Silva, Floriano P

    2016-06-01

    The use of computational filters for excluding supposedly nonspecific and promiscuous compounds from chemical libraries is a controversial issue, because many drugs used in clinics today would never reach the market if these filters were applied. In part, this conflict could be caused by the paradigm: one-drug-one-target, even though it is widely agreed that drug action is a result of a complex network of biomolecular interactions. Therefore, the so-called pan assay interference compounds (PAINS) or promiscuous compounds could be in fact assay artifacts, false positives or, simply, bright chemical matter (BCM) composed of privileged scaffolds, as we propose here. Despite apparent promiscuity, BCM can be tailored into new and safe drugs after overcoming selectivity criteria. PMID:26880580

  5. A cell-signaling network temporally resolves specific versus promiscuous phosphorylation.

    PubMed

    Kanshin, Evgeny; Bergeron-Sandoval, Louis-Philippe; Isik, S Sinan; Thibault, Pierre; Michnick, Stephen W

    2015-02-24

    If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG) response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis. PMID:25704821

  6. Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis.

    PubMed

    Jia, Meirong; Potter, Kevin C; Peters, Reuben J

    2016-09-01

    Diterpenes are widely distributed across many biological kingdoms, where they serve a diverse range of physiological functions, and some have significant industrial utility. Their biosynthesis involves class I diterpene synthases (DTSs), whose activity can be preceded by that of class II diterpene cyclases (DTCs). Here, a modular metabolic engineering system was used to examine the promiscuity of DTSs. Strikingly, both a bacterial and plant DTS were found to exhibit extreme promiscuity, reacting with all available precursors with orthogonal activity, producing an olefin or hydroxyl group, respectively. Such DTS promiscuity enables combinatorial biosynthesis, with remarkably high yields for these unoptimized non-native enzymatic combinations (up to 15mg/L). Indeed, it was possible to readily characterize the 13 unknown products. Notably, 16 of the observed diterpenes were previously inaccessible, and these results provide biosynthetic routes that are further expected to enable assembly of more extended pathways to produce additionally elaborated 'non-natural' diterpenoids. PMID:27060773

  7. The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase.

    PubMed

    Bredeston, Luis M; González Flecha, F Luis

    2016-07-01

    Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP. PMID:27086711

  8. Proton-in-Flight Mechanism for the Spontaneous Hydrolysis of N-Methyl O-Phenyl Sulfamate: Implications for the Design of Steroid Sulfatase Inhibitors

    PubMed Central

    Edwards, David R.; Wolfenden, Richard

    2012-01-01

    The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 1011. Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with moderate efficiencies of ~104; whereas, the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~1015. Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO2X−, are shown to be correlated by a two parameter equation based on pKaArOH and pKaArOSO2XH. PMID:22486328

  9. The "Promiscuous Audience" Controversy and the Emergence of the Early Woman's Rights Movement.

    ERIC Educational Resources Information Center

    Zaeske, Susan

    1995-01-01

    Examines the "Promiscuous Audience" charge against activist women in the 1830s--its emergence, persuasive force, motivations, and responses to it. Shows how, in establishing their right to speak from public platforms, activist women did not rely on natural law or Constitutional appeals, but rather emphasized the special nature of female…

  10. Broadening the Concept of Adolescent Promiscuity: Male Accountability Made Visible and the Implications for Family Therapists.

    ERIC Educational Resources Information Center

    Dankoski, Mary E.; Payer, Rosemary; Steinberg, Marilyn

    1996-01-01

    Looks at male promiscuity and the gender bias that holds females accountable for sexual activity. Examines communication patterns, family structure, and other factors. Argues that family therapists can alter family communication patterns, redefine boundaries, and promote healthy parental involvement to make an impact on the issue of male sexual…

  11. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  12. Predictors of Drug/Alcohol Abuse and Sexual Promiscuity of College Students.

    ERIC Educational Resources Information Center

    Nam, Jeong Sook; And Others

    This study examined the relationship between the individual's purpose in life, existential anxiety, powerlessness and use of alcohol/drugs and the tendency to be sexually promiscuous. The study is rooted in the work of Viktor E. Frankl, which suggested that a lack of meaning and purpose can cause socially deviant behavior and psychological…

  13. Monogamous and promiscuous rodent species exhibit discrete variation in the size of the medial prefrontal cortex.

    PubMed

    Kingsbury, Marcy A; Gleason, Erin D; Ophir, Alexander G; Phelps, Steven M; Young, Larry J; Marler, Catherine A

    2012-01-01

    Limbic-associated cortical areas, such as the medial prefrontal and retrosplenial cortex (mPFC and RS, respectively), are involved in the processing of emotion, motivation, and various aspects of working memory and have been implicated in mating behavior. To determine whether the independent evolution of mating systems is associated with a convergence in cortical mechanisms, we compared the size of mPFC and RS between the monogamous prairie vole (Microtus ochrogaster) and the promiscuous meadow vole (Microtus pennsylvanicus), and between the monogamous California mouse (Peromyscus californicus) and the promiscuous white-footed mouse (Peromyscus leucopus). For both promiscuous mice and voles, the mPFC occupied a significantly larger percentage of total cortex than in the monogamous species. No significant differences were observed for the RS or overall cortex size with respect to mating system, supporting the convergent evolution of mPFC size, specifically. Individual differences in the mating behavior of male prairie voles (wandering versus pair-bonding), presumably facultative tactics, were not reflected in the relative size of the mPFC, which is likely a heritable trait. Given the importance of the mPFC for complex working memory, particularly object-place and temporal order memory, we hypothesize that the relatively greater size of the mPFC in promiscuous species reflects a greater need to remember multiple individuals and the times and locations in which they have been encountered in the home range. PMID:22759599

  14. Steroid sulfatase inhibitor DU-14 protects spatial memory and synaptic plasticity from disruption by amyloid β protein in male rats.

    PubMed

    Yue, Xing-Hua; Tong, Jia-Qing; Wang, Zhao-Jun; Zhang, Jun; Liu, Xu; Liu, Xiao-Jie; Cai, Hong-Yan; Qi, Jin-Shun

    2016-07-01

    Alzheimer's disease (AD) is an age-related mental disorder characterized by progressive loss of memory and multiple cognitive impairments. The overproduction and aggregation of Amyloid β protein (Aβ) in the brain, especially in the hippocampus, are closely involved in the memory loss in the patients with AD. Accumulating evidence indicates that the Aβ-induced imbalance of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) in the brain plays an important role in the AD pathogenesis and progression. The level of DHEA is elevated, while DHEAS is dramatically decreased in the AD brain. The present study tried to restore the balance between DHEA and DHEAS by using a non-steroidal sulfatase inhibitor DU-14, which increases endogenous DHEAS through preventing DHEAS converted back into DHEA. We found that: (1) DU-14 effectively attenuated the Aβ1-42-induced cognitive deficits in spatial learning and memory of rats in Morris water maze test; (2) DU-14 prevented Aβ1-42-induced decrease in the cholinergic theta rhythm of hippocampal local field potential (LFP) in the CA1 region; (3) DU-14 protected hippocampal synaptic plasticity against Aβ1-42-induced suppression of long term potentiation (LTP). These results provide evidence for the neuroprotective action of DU-14 against neurotoxic Aβ, suggesting that up-regulation of endogenous DHEAS by DU-14 could be beneficial to the alleviation of Aβ-induced impairments in spatial memory and synaptic plasticity. PMID:27222435

  15. The Effect of Recombinant Human Iduronate-2-Sulfatase (Idursulfase) on Growth in Young Patients with Mucopolysaccharidosis Type II

    PubMed Central

    Żuber, Zbigniew; Różdżyńska-Świątkowska, Agnieszka; Jurecka, Agnieszka; Tylki-Szymańska, Anna

    2014-01-01

    Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is an X-linked, recessive, lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase. Early bone involvement leads to decreased growth velocity and short stature in nearly all patients. Our analysis aimed to investigate the effects of enzyme replacement therapy (ERT) with idursulfase (Elaprase) on growth in young patients with mucopolysaccharidosis type II. Analysis of longitudinal anthropometric data of MPS II patients (group 1, n = 13) who started ERT before 6 years of age (range from 3 months to 6 years, mean 3.6 years, median 4 years) was performed and then compared with retrospective analysis of data for MPS II patients naïve to ERT (group 2, n = 50). Patients in group 1 received intravenous idursulfase at a standard dose of 0.58 mg/kg weekly for 52–288 weeks. The course of average growth curve for group 1 was very similar to growth pattern in group 2. The average value of body height in subsequent years in group 1 was a little greater than in group 2, however, the difference was not statistically significant. In studied patients with MPS II, idursulfase did not appear to alter the growth patterns. PMID:24454794

  16. Design, synthesis, and biological evaluation of new arylamide derivatives possessing sulfonate or sulfamate moieties as steroid sulfatase enzyme inhibitors.

    PubMed

    El-Gamal, Mohammed I; Semreen, Mohammad H; Foster, Paul A; Potter, Barry V L

    2016-06-15

    A series of new arylamide derivatives possessing terminal sulfonate or sulfamate moieties was designed and synthesized. The target compounds were tested for in vitro inhibitory effects against the steroid sulfatase (STS) enzyme in a cell-free assay system. The free sulfamate derivative 1j was the most active. It inhibited the enzymatic activity by 72.0% and 55.7% at 20μM and 10μM, respectively. Compound 1j was further tested for STS inhibition in JEG-3 placental carcinoma cells with high STS enzyme activity. It inhibited 93.9% of the enzyme activity in JEG-3 placental carcinoma cells at 20μM with an efficacy near to that of the well-established drug STX64 as reference. At 10μM, 1j inhibited 86.1% of the STS activity of JEG-3. Its IC50 value against the STS enzyme in JEG-3 cells was 0.421μM. Thus, 1j represents an attractive new non-steroidal lead for further optimization. PMID:27143133

  17. The Effect of The Steroid Sulfatase Inhibitor (p-O-sulfamoyl)–Tetradecanoyl Tyramine (DU-14) on Learning and Memory in Rats with Selective Lesion of Septal-Hippocampal Cholinergic Tract

    PubMed Central

    Babalola, P. A.; Fitz, N.F.; Gibbs, R.B; Flaherty, P.; Li, P.-K.; Johnson, D.A.

    2012-01-01

    Dehydroepiandrosterone sulfate (DHEAS), is an excitatory neurosteroid synthesized within the CNS that modulates brain function. Effects associated with augmented DHEAS include learning and memory enhancement. Inhibitors of the steroid sulfatase enzyme increase brain DHEAS levels and can also facilitate learning and memory. This study investigated the effect of steroid sulfatase inhibition on learning and memory in rats with selective cholinergic lesion of the septo-hippocampal tract using passive avoidance and delayed matching to position T-maze (DMP) paradigms. The selective cholinergic immunotoxin 192 IgG-saporin (SAP) was infused into the medial septum of animals and then tested using a step-through passive avoidance paradigm or DMP paradigm. Peripheral administration of the steroid sulfatase inhibitor, DU-14, increased step-through latency following footshock in rats with SAP lesion compared to both vehicle treated control and lesioned animals (p < 0.05). However, in the DMP task, steroid sulfatase inhibition impaired acquisition in lesioned rats while having no effect on intact animals. These results suggest that steroid sulfatase inhibition facilitates memory associated with contextual fear, but impairs acquisition of spatial memory tasks in rats with selective lesion of the septo-hippocampal tract. PMID:23022361

  18. Z linkage of female promiscuity genes in the moth Utetheisa ornatrix: support for the sexy-sperm hypothesis?

    PubMed

    Iyengar, Vikram K; Reeve, Hudson K

    2010-05-01

    Female preference genes for large males in the highly promiscuous moth Utetheisa ornatrix (Lepidoptera: Arctiidae) have previously been shown to be mostly Z-linked, in accordance with the hypothesis that ZZ-ZW sex chromosome systems should facilitate Fisherian sexual selection. We determined the heritability of both female and male promiscuity in the highly promiscuous moth U. ornatrix (Lepidoptera: Arctiidae) through parent-offspring and grandparent-offspring regression analyses. Our data show that male promiscuity is not sex-limited and either autosomal or sex-linked whereas female promiscuity is primarily determined by sex-limited, Z-linked genes. These data are consistent with the "sexy-sperm hypothesis," which posits that multiple-mating and sperm competitiveness coevolve through a Fisherian-like process in which female promiscuity is a kind of mate choice in which sperm-competitiveness is the trait favored in males. Such a Fisherian process should also be more potent when female preferences are Z-linked and sex-limited than when autosomal or not limited. PMID:20002164

  19. Substrate Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans

    PubMed Central

    Cohen-Rosenzweig, Chen; Guan, Ziqiang; Shaanan, Boaz

    2014-01-01

    Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. PMID:24212570

  20. Substrate promiscuity: AglB, the archaeal oligosaccharyltransferase, can process a variety of lipid-linked glycans.

    PubMed

    Cohen-Rosenzweig, Chen; Guan, Ziqiang; Shaanan, Boaz; Eichler, Jerry

    2014-01-01

    Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. PMID:24212570

  1. Molecular analysis of paternity shows promiscuous mating in female humpback whales (Megaptera novaeangliae, Borowski).

    PubMed Central

    Clapham, P J; Palsbøll, P J

    1997-01-01

    It is widely assumed that the mating system of the humpback whale. Magaptera novaeangliae, is similar to that of most mammals in that it represents some form of polygyny or promiscuity, but this cannot be tested without observations of copulation or data on paternity of offspring. Microsatellite DNA markers were used to examine the paternity of calves born to individually identified mature female humpback whales from the Gulf of Maine. Skin biopsies were obtained from three females, and several (range: three to five) of their known offspring. Multiple paternity of offspring, indicated by the presence of at least three different paternal alleles, was evident in all three females at either three or four of the six microsatellite loci surveyed. Such promiscuous mating is expected given current knowledge of the social ecology of this species. It is also consistent with resightings of individually identified female humpbacks with different male associates during two or more breeding seasons. PMID:9061965

  2. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

    PubMed Central

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte M.; Fischer, Gerhard; Mohamed, Mark F.; Hyvönen, Marko; Morgavi, Diego P.; Janssen, Dick B; Hollfelder, Florian

    2015-01-01

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions. PMID:26639611

  3. Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

    SciTech Connect

    Gupta,Y.; Nair, D.; Wharton, R.; Aggarwal, A.

    2008-01-01

    Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a 'spacer.' The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.

  4. The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

    PubMed Central

    Münz, Márton; Hein, Jotun; Biggin, Philip C.

    2012-01-01

    In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

  5. Differential plant invasiveness is not always driven by host promiscuity with bacterial symbionts.

    PubMed

    Klock, Metha M; Barrett, Luke G; Thrall, Peter H; Harms, Kyle E

    2016-01-01

    Identification of mechanisms that allow some species to outcompete others is a fundamental goal in ecology and invasive species management. One useful approach is to examine congeners varying in invasiveness in a comparative framework across native and invaded ranges. Acacia species have been widely introduced outside their native range of Australia, and a subset of these species have become invasive in multiple parts of the world. Within specific regions, the invasive status of these species varies. Our study examined whether a key mechanism in the life history of Acacia species, the legume-rhizobia symbiosis, influences acacia invasiveness on a regional scale. To assess the extent to which species varying in invasiveness correspondingly differ with regard to the diversity of rhizobia they associate with, we grew seven Acacia species ranging in invasiveness in California in multiple soils from both their native (Australia) and introduced (California) ranges. In particular, the aim was to determine whether more invasive species formed symbioses with a wider diversity of rhizobial strains (i.e. are more promiscuous hosts). We measured and compared plant performance, including aboveground biomass, survival, and nodulation response, as well as rhizobial community composition and richness. Host promiscuity did not differ among invasiveness categories. Acacia species that varied in invasiveness differed in aboveground biomass for only one soil and did not differ in survival or nodulation within individual soils. In addition, acacias did not differ in rhizobial richness among invasiveness categories. However, nodulation differed between regions and was generally higher in the native than introduced range. Our results suggest that all Acacia species introduced to California are promiscuous hosts and that host promiscuity per se does not explain the observed differences in invasiveness within this region. Our study also highlights the utility of assessing potential

  6. Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases.

    PubMed

    Hou, Liming; Honaker, Matthew T; Shireman, Laura M; Balogh, Larissa M; Roberts, Arthur G; Ng, Kei-Cheuk; Nath, Abhinav; Atkins, William M

    2007-08-10

    The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability. PMID:17561509

  7. Differential plant invasiveness is not always driven by host promiscuity with bacterial symbionts

    PubMed Central

    Klock, Metha M.; Barrett, Luke G.; Thrall, Peter H.; Harms, Kyle E.

    2016-01-01

    Identification of mechanisms that allow some species to outcompete others is a fundamental goal in ecology and invasive species management. One useful approach is to examine congeners varying in invasiveness in a comparative framework across native and invaded ranges. Acacia species have been widely introduced outside their native range of Australia, and a subset of these species have become invasive in multiple parts of the world. Within specific regions, the invasive status of these species varies. Our study examined whether a key mechanism in the life history of Acacia species, the legume-rhizobia symbiosis, influences acacia invasiveness on a regional scale. To assess the extent to which species varying in invasiveness correspondingly differ with regard to the diversity of rhizobia they associate with, we grew seven Acacia species ranging in invasiveness in California in multiple soils from both their native (Australia) and introduced (California) ranges. In particular, the aim was to determine whether more invasive species formed symbioses with a wider diversity of rhizobial strains (i.e. are more promiscuous hosts). We measured and compared plant performance, including aboveground biomass, survival, and nodulation response, as well as rhizobial community composition and richness. Host promiscuity did not differ among invasiveness categories. Acacia species that varied in invasiveness differed in aboveground biomass for only one soil and did not differ in survival or nodulation within individual soils. In addition, acacias did not differ in rhizobial richness among invasiveness categories. However, nodulation differed between regions and was generally higher in the native than introduced range. Our results suggest that all Acacia species introduced to California are promiscuous hosts and that host promiscuity per se does not explain the observed differences in invasiveness within this region. Our study also highlights the utility of assessing potential

  8. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens

    PubMed Central

    Caro-Aguilar, Ivette; Rodríguez, Alexandra; Calvo-Calle, J. Mauricio; Guzmán, Fanny; De la Vega, Patricia; Elkin Patarroyo, Manuel; Galinski, Mary R.; Moreno, Alberto

    2002-01-01

    Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development. PMID:12065487

  9. An ancient but promiscuous host-symbiont association between Burkholderia gut symbionts and their heteropteran hosts.

    PubMed

    Kikuchi, Yoshitomo; Hosokawa, Takahiro; Fukatsu, Takema

    2011-03-01

    Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host-symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host-symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host-symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed. PMID:20882057

  10. The Promiscuity of Allosteric Regulation of Nuclear Receptors by Retinoid X Receptor.

    PubMed

    Clark, Alexander K; Wilder, J Heath; Grayson, Aaron W; Johnson, Quentin R; Lindsay, Richard J; Nellas, Ricky B; Fernandez, Elias J; Shen, Tongye

    2016-08-25

    The promiscuous protein retinoid X receptor (RXR) displays essential allosteric regulation of several members in the nuclear hormone receptor superfamily via heterodimerization and (anti)cooperative binding of cognate ligands. Here, the structural basis of the positive allostery of RXR and constitutive androstane receptor (CAR) is revealed. In contrast, a similar computational approach had previously revealed the mechanism for negative allostery in the complex of RXR and thyroid receptor (TR). By comparing the positive and negative allostery of RXR complexed with CAR and TR respectively, we reported the promiscuous allosteric control involving RXR. We characterize the allosteric mechanism by expressing the correlated dynamics of selected residue-residue contacts which was extracted from atomistic molecular dynamics simulation and statistical analysis. While the same set of residues in the binding pocket of RXR may initiate the residue-residue interaction network, RXR uses largely different sets of contacts (only about one-third identical) and allosteric modes to regulate TR and CAR. The promiscuity of RXR control may originate from multiple factors, including (1) the frustrated fit of cognate ligand 9c to the RXR binding pocket and (2) the different ligand-binding features of TR (loose) versus CAR (tight) to their corresponding cognate ligands. PMID:27110634

  11. Steroid signaling: ligand-binding promiscuity, molecular symmetry, and the need for gating.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri

    2014-04-01

    Steroid/sterol-binding receptors and enzymes are remarkably promiscuous in the range of ligands they can bind to and, in the case of enzymes, modify - raising the question of how specific receptor activation is achieved in vivo. Estrogen receptors (ER) are modulated by 27-hydroxycholesterol and 5α-androstane-3β,17β-diol (Adiol), in addition to estradiol (E2), and respond to diverse small molecules such as bisphenol A. Steroid-modifying enzymes are also highly promiscuous in ligand binding and metabolism. The specificity problem is compounded by the fact that the steroid core (hydrogenated cyclopentophenanthrene ring system) has several planes of symmetry. Ligand binding can be in symmetrical East-West (rotation) and North-South (inversion) orientations. Hydroxysteroid dehydrogenases (HSDs) can modify symmetrical 7 and 11, also 3 and 17/20, positions, exemplified here by yeast 3α,20β-HSD and mammalian 11β-HSD and 17β-HSD enzymes. Faced with promiscuity and symmetry, other strategies are clearly necessary to promote signaling selectivity in vivo. Gating regulates hormone access via enzymes that preferentially inactivate (or activate) a subclass of ligands, thereby governing which ligands gain receptor access - exemplified by 11β-HSD gating cortisol access to the mineralocorticoid receptor, and P450 CYP7B1 gating Adiol access to ER. Counter-intuitively, the specificity of steroid/sterol action is achieved not by intrinsic binding selectivity but by the combination of local metabolism and binding affinity. PMID:24462647

  12. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  13. Induction of steroid sulfatase expression in PC-3 human prostate cancer cells by insulin-like growth factor II.

    PubMed

    Sung, Chul-Hoon; Im, Hee-Jung; Park, Nahee; Kwon, Yeojung; Shin, Sangyun; Ye, Dong-Jin; Cho, Nam-Hyeon; Park, Young-Shin; Choi, Hyung-Kyoon; Kim, Donghak; Chun, Young-Jin

    2013-11-25

    Human steroid sulfatase (STS) plays an important role in regulating the formation of biologically active estrogens and may be a promising target for treating estrogen-mediated carcinogenesis. The molecular mechanism of STS gene expression, however, is still not clear. Growth factors are known to increase STS activity but the changes in STS expression have not been completely understood. To determine whether insulin-like growth factor (IGF)-II can induce STS gene expression, the effects of IGF-II on STS expression were studied in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that IGF-II treatment significantly increased the expression of STS mRNA and protein in concentration- and time-dependent manners. To understand the signaling pathway by which IGF-II induces STS gene expression, the effects of specific PI3-kinase/Akt and NF-κB inhibitors were determined. When the cells were treated with IGF-II and PI3-kinase/Akt inhibitors, such as LY294002, wortmannin, or Akt inhibitor IV, STS expression induced by IGF-II was significantly blocked. Moreover, we found that NF-κB inhibitors, such as MG-132, bortezomib, Bay 11-7082 or Nemo binding domain (NBD) binding peptide, also strongly prevented IGF-II from inducing STS gene expression. We assessed whether IGF-II activates STS promoter activity using transient transfection with a luciferase reporter. IGF-II significantly stimulated STS reporter activity. Furthermore, IGF-II induced expression of 17β-hydroxysteroid dehydrogenase (HSD) 1 and 3, whereas it reduced estrone sulfotransferase (EST) gene expression, causing enhanced estrone and β-estradiol production. Taken together, these results strongly suggest that IGF-II induces STS expression via a PI3-kinase/Akt-NF-κB signaling pathway in PC-3 cells and may induce estrogen production and estrogen-mediated carcinogenesis. PMID:24055520

  14. Effects of steroid hormone on estrogen sulfotransferase and on steroid sulfatase expression in endometriosis tissue and stromal cells.

    PubMed

    Piccinato, Carla A; Neme, Rosa M; Torres, Natália; Sanches, Lívia Renta; Derogis, Priscilla Bento Mattos Cruz; Brudniewski, Heloísa F; Rosa e Silva, Júlio C; Ferriani, Rui A

    2016-04-01

    Endometriosis is an estrogen-dependent disease that afflicts about 10% of women in their reproductive age, causing severe pain and infertility. The potential roles of female steroid hormones in modulating key estrogen-metabolizing enzymes, steroid sulfatase (STS) and estrogen sulfotransferase (SULT1E1), were investigated. The expression of STS and SULT1E1 mRNA in biopsy samples (n=78) of superficial and deep endometriotic lesions, eutopic endometrium of women with endometriosis and endometrium from control patients were compared according to the menstrual cycle phase. Increased STS gene expression was detected in superficial and deep-infiltrating lesions and a reduced SULT1E1 expression was also observed in the eutopic endometrium relative to the superficial lesions. Additionally, a significantly positive correlation was detected between STS and SULT1E1 mRNA expression levels in biopsy specimens collected from the endometriosis patients, and not in control individuals. The actions of female steroid hormones on SULT1E1 and STS expression were evidenced in endometriosis, revealed by increased expression levels in the luteal phase of the cycle. There was an increased STS expression in primary eutopic and ectopic endometrial stromal cells treated with estradiol and progesterone (representative of the luteal phase, n=3). Although an increased STS mRNA expression was observed in hormone-induced endometrial stromal cells in vitro, no difference could be detected between the hormone treatment groups in estradiol formation from estradiol sulfate measured by LC-MS-MS. Interestingly, a greater expression of STS was observed in stromal cells from eutopic endometrium with an agreement in estradiol formation originated from estradiol sulfate. The differential regulation of STS and SULT1E1 could provide insights for novel studies of the therapeutic use of STS inhibitors. PMID:26723541

  15. A study of the relationship between clinical phenotypes and plasma iduronate-2-sulfatase enzyme activities in Hunter syndrome patients

    PubMed Central

    Lee, Ok Jeong; Kim, Su-Jin; Sohn, Young Bae; Park, Hyung-Doo; Lee, Soo-Youn; Kim, Chi-Hwa; Ko, Ah-Ra; Yook, Yeon-Joo; Lee, Su-Jin; Park, Sung Won; Kim, Se-Hwa; Cho, Sung-Yoon; Kwon, Eun-Kyung; Han, Sun Ju

    2012-01-01

    Purpose Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a rare lysosomal storage disorder caused by iduronate-2-sulfatase (IDS) deficiency. MPS II causes a wide phenotypic spectrum of symptoms ranging from mild to severe. IDS activity, which is measured in leukocyte pellets or fibroblasts, was reported to be related to clinical phenotype by Sukegawa-Hayasaka et al. Measurement of residual plasma IDS activity using a fluorometric assay is simpler than conventional measurements using skin fibroblasts or peripheral blood mononuclear cells. This is the first study to describe the relationship between plasma IDS activity and clinical phenotype of MPS II. Methods We hypothesized that residual plasma IDS activity is related to clinical phenotype. We classified 43 Hunter syndrome patients as having attenuated or severe disease types based on clinical characteristics, especially intellectual and cognitive status. There were 27 patients with the severe type and 16 with the attenuated type. Plasma IDS activity was measured by a fluorometric enzyme assay using 4-methylumbelliferyl-α-iduronate 2-sulphate. Results Plasma IDS activity in patients with the severe type was significantly lower than that in patients with the attenuated type (P=0.006). The optimal cut-off value of plasma IDS activity for distinguishing the severe type from the attenuated type was 0.63 nmol·4 hr-1·mL-1. This value had 88.2% sensitivity, 65.4% specificity, and an area under receiver-operator characteristics (ROC) curve of 0.768 (ROC curve analysis; P=0.003). Conclusion These results show that the mild phenotype may be related to residual lysosomal enzyme activity. PMID:22474463

  16. Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin

    PubMed Central

    Kotlo, Kumar; Bhattacharyya, Sumit; Yang, Bo; Feferman, Leonid; Tejaskumar, Shah; Linhardt, Robert; Danziger, Robert

    2013-01-01

    N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation. PMID:23385884

  17. Hepatic Overexpression of Steroid Sulfatase Ameliorates Mouse Models of Obesity and Type 2 Diabetes through Sex-specific Mechanisms*

    PubMed Central

    Jiang, Mengxi; He, Jinhan; Kucera, Heidi; Gaikwad, Nilesh W.; Zhang, Bin; Xu, Meishu; O'Doherty, Robert M.; Selcer, Kyle W.; Xie, Wen

    2014-01-01

    The steroid sulfatase (STS)-mediated desulfation is a critical metabolic mechanism that regulates the chemical and functional homeostasis of endogenous and exogenous molecules. In this report, we first showed that the liver expression of Sts was induced in both the high fat diet (HFD) and ob/ob models of obesity and type 2 diabetes and during the fed to fasting transition. In defining the functional relevance of STS induction in metabolic disease, we showed that overexpression of STS in the liver of transgenic mice alleviated HFD and ob/ob models of obesity and type 2 diabetes, including reduced body weight, improved insulin sensitivity, and decreased hepatic steatosis and inflammation. Interestingly, STS exerted its metabolic benefit through sex-specific mechanisms. In female mice, STS may have increased hepatic estrogen activity by converting biologically inactive estrogen sulfates to active estrogens and consequently improved the metabolic functions, whereas ovariectomy abolished this protective effect. In contrast, the metabolic benefit of STS in males may have been accounted for by the male-specific decrease of inflammation in white adipose tissue and skeletal muscle as well as a pattern of skeletal muscle gene expression that favors energy expenditure. The metabolic benefit in male STS transgenic mice was retained after castration. Treatment with the STS substrate estrone sulfate also improved metabolic functions in both the HFD and ob/ob models. Our results have uncovered a novel function of STS in energy metabolism and type 2 diabetes. Liver-specific STS induction or estrogen/estrogen sulfate delivery may represent a novel approach to manage metabolic syndrome. PMID:24497646

  18. Heteroallelic missense mutations of the galactosamine-6-sulfate sulfatase (GALNS) gene in a mild form of Morquio disease (MPS IVA)

    SciTech Connect

    Cole, D.E.C.; Gordon, B.A.; Rupar, C.A.

    1996-06-28

    Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 {plus_minus} 5% of controls (as compared to 5 {plus_minus} 3% of controls in severe MPS IVA, P <.003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K{sub m} and increased V{sub max} in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mile GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines. 30 refs., 4 figs., 3 tabs.

  19. Theoretical Proposal for the Whole Phosphate Diester Hydrolysis Mechanism Promoted by a Catalytic Promiscuous Dinuclear Copper(II) Complex.

    PubMed

    Esteves, Lucas F; Rey, Nicolás A; Dos Santos, Hélio F; Costa, Luiz Antônio S

    2016-03-21

    The catalytic mechanism that involves the cleavage of the phosphate diester model BDNPP (bis(2,4-dinitrophenyl) phosphate) catalyzed through a dinuclear copper complex is investigated in the current study. The metal complex was originally designed to catalyze catechol oxidation, and it showed an interesting catalytic promiscuity case in biomimetic systems. The current study investigates two different reaction mechanisms through quantum mechanics calculations in the gas phase, and it also includes the solvent effect through PCM (polarizable continuum model) single-point calculations using water as solvent. Two mechanisms are presented in order to fully describe the phosphate diester hydrolysis. Mechanism 1 is of the SN2 type, which involves the direct attack of the μ-OH bridge between the two copper(II) ions toward the phosphorus center, whereas mechanism 2 is the process in which hydrolysis takes place through proton transfer between the oxygen atom in the bridging hydroxo ligand and the other oxygen atom in the phosphate model. Actually, the present theoretical study shows two possible reaction paths in mechanism 1. Its first reaction path (p1) involves a proton transfer that occurs immediately after the hydrolytic cleavage, so that the proton transfer is the rate-determining step, which is followed by the entry of two water molecules. Its second reaction path (p2) consists of the entry of two water molecules right after the hydrolytic cleavage, but with no proton transfer; thus, hydrolytic cleavage is the rate-limiting step. The most likely catalytic path occurs in mechanism 1, following the second reaction path (p2), since it involves the lowest free energy activation barrier (ΔG(⧧) = 23.7 kcal mol(-1), in aqueous solution). A kinetic analysis showed that the experimental kobs value of 1.7 × 10(-5) s(-1) agrees with the calculated value k1 = 2.6 × 10(-5) s(-1); the concerted mechanism is kinetically favorable. The KIE (kinetic isotope effect) analysis

  20. The Role of Heparanase and Sulfatases in the Modification of Heparan Sulfate Proteoglycans within the Tumor Microenvironment and Opportunities for Novel Cancer Therapeutics

    PubMed Central

    Hammond, Edward; Khurana, Ashwani; Shridhar, Viji; Dredge, Keith

    2014-01-01

    Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix. The modification of HSPGs in the tumor microenvironment is known to result not just in structural but also functional consequences, which significantly impact cancer progression. As substrates for the key enzymes sulfatases and heparanase, the modification of HSPGs is typically characterized by the degradation of heparan sulfate (HS) chains/sulfation patterns via the endo-6-O-sulfatases (Sulf1 and Sulf2) or by heparanase, an endo-glycosidase that cleaves the HS polymers releasing smaller fragments from HSPG complexes. Numerous studies have demonstrated how these enzymes actively influence cancer cell proliferation, signaling, invasion, and metastasis. The activity or expression of these enzymes has been reported to be modified in a variety of cancers. Such observations are consistent with the degradation of normal architecture and basement membranes, which are typically compromised in metastatic disease. Moreover, recent studies elucidating the requirements for these proteins in tumor initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumor microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review discusses the role of these enzymes in the context of the tumor microenvironment and their promise as therapeutic targets for the treatment of cancer. PMID:25105093

  1. Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

    NASA Astrophysics Data System (ADS)

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-10-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions.

  2. Protease-catalysed direct asymmetric Mannich reaction in organic solvent.

    PubMed

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-01-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions. PMID:23094136

  3. Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

    PubMed Central

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-01-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions. PMID:23094136

  4. Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB.

    PubMed

    Kronen, Miriam; Berg, Ivan A

    2015-01-01

    Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency kcat/Km, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration. PMID:26658641

  5. Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

    PubMed Central

    Kronen, Miriam; Berg, Ivan A.

    2015-01-01

    Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency kcat/Km, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration. PMID:26658641

  6. Reporting Sodium Channel Activity Using Calcium Flux: Pharmacological Promiscuity of Cardiac Nav1.5

    PubMed Central

    Zhang, Hongkang; Zou, Beiyan; Du, Fang; Xu, Kaiping

    2015-01-01

    Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 μM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions. PMID:25422141

  7. Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian β-lactamases.

    PubMed

    Risso, Valeria A; Gavira, Jose A; Mejia-Carmona, Diego F; Gaucher, Eric A; Sanchez-Ruiz, Jose M

    2013-02-27

    We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution. PMID:23394108

  8. The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms

    PubMed Central

    2013-01-01

    Background ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate. Results The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP. Conclusions After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs. PMID:23433303

  9. Promiscuity and pill: etiologic agents in the genesis of cervical malignancy.

    PubMed

    Leppäluoto, P A

    1977-01-01

    An 800% increase in the number of "suspicious" Papanicolaou smears from young women have been reported since 1970. In cases diagnosed from tissue specimens as "surface dysplasia," the majority of patients gave histories of use of birth control pills and sexual promiscuity. Sexually active women have an increased incidence of cervical ectopy. In cervical ectopy the endocervical columnar cells are exposed to the acid vaginal content. Development of squamous metaplasia may result. An ideal contraceptive would be one contributing to the preservation of a healthy cervical epithelium without ectopy, and a healthy vaginal content as shown by the presence of Doderlein flora. These conditions would serve to prevent cervical malignancy. PMID:266326

  10. Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

    PubMed Central

    Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

    2016-01-01

    As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

  11. Characterizing the Promiscuity of LigAB, a Lignin Catabolite Degrading Extradiol Dioxygenase from Sphingomonas paucimobilis SYK-6

    PubMed Central

    Barry, Kevin P.; Taylor, Erika A.

    2014-01-01

    LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s−1 and a kcat/KM of 4.26 × 106 M−1s−1. LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ~4-fold lower than that for gallate and ~10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically. PMID:23977959

  12. Model studies on the first enzyme-catalyzed Ugi reaction.

    PubMed

    Kłossowski, Szymon; Wiraszka, Barbara; Berłożecki, Stanisław; Ostaszewski, Ryszard

    2013-02-01

    Multicomponent reactions are powerful tools for organic chemistry, and among them, the Ugi reaction provides remarkable improvement in many fields of organic chemistry such us combinatorial chemistry, medicinal chemistry, and peptide chemistry. A new, enzyme-catalyzed example of the Ugi three-component reaction is presented. The studies include the selection of an enzyme as well as determination of the scope and limitations of the newly described reaction. The presented method combines the enzyme promiscuity and multicomponent reaction advantages in the first one-pot formation of dipeptide 1. PMID:23343100

  13. QM/MM analysis suggests that Alkaline Phosphatase (AP) and Nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily

    PubMed Central

    Hou, Guanhua

    2011-01-01

    Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proficiency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP−, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parameterized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semi-quantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and coworkers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters

  14. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    PubMed

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. PMID:26682704

  15. The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

    PubMed Central

    Ganesan, Lakshmi; Buchwald, Peter

    2013-01-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

  16. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  17. Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

    SciTech Connect

    Miyagawa, Atsushi; Totani, Kiichiro; Matsuo, Ichiro; Ito, Yukishige

    2010-12-17

    Research highlights: {yields} UGGT has a narrow donor specificity. {yields} UGGT gave several non-natural high-mannose-type glycans. {yields} G-II has a promiscuous activity as broad specificity hexosidase. -- Abstract: In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man{sub 9}GlcNAc{sub 2}, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

  18. Optical state engineering, quantum communication, and robustness of entanglement promiscuity in three-mode Gaussian states

    NASA Astrophysics Data System (ADS)

    Adesso, Gerardo; Serafini, Alessio; Illuminati, Fabrizio

    2007-03-01

    We present a novel, detailed study on the usefulness of three-mode Gaussian states for realistic processing of continuous variable (CV) quantum information, with a particular emphasis on the possibilities opened up by their genuine tripartite entanglement. We describe practical schemes to engineer several classes of pure and mixed three-mode states that stand out for their informational and/or entanglement properties. In particular, we introduce a simple procedure—based on passive optical elements—to produce pure three-mode Gaussian states with arbitrary entanglement structure (upon availability of an initial two-mode squeezed state). We analyse in depth the properties of distributed entanglement and the origin of its sharing structure, showing that the promiscuity of entanglement sharing is a feature peculiar to symmetric Gaussian states that survives even in the presence of significant degrees of mixedness and decoherence. Next, we discuss the suitability of the considered tripartite entangled states to the implementation of quantum information and communication protocols with CVs. This will lead to a feasible experimental proposal to test the promiscuous sharing of CV tripartite entanglement, in terms of the optimal fidelity of teleportation networks with Gaussian resources. We finally focus on the application of three-mode states to symmetric and asymmetric telecloning, and single out the structural properties of the optimal Gaussian resources for the latter protocol in different settings. Our analysis aims to lay the basis for a practical quantum communication with CVs beyond the bipartite scenario.

  19. Privileged scaffolds or promiscuous binders: a comparative study on rhodanines and related heterocycles in medicinal chemistry.

    PubMed

    Mendgen, Thomas; Steuer, Christian; Klein, Christian D

    2012-01-26

    Rhodanines and related five-membered heterocycles with multiple heteroatoms have recently gained a reputation of being unselective compounds that appear as "frequent hitters" in screening campaigns and therefore have little value in drug discovery. However, this judgment appears to be based mostly on anecdotal evidence. Having identified various rhodanines and related compounds in screening campaigns, we decided to perform a systematic study on their promiscuity. An amount of 163 rhodanines, hydantoins, thiohydantoins, and thiazolidinediones were synthesized and tested against several targets. The compounds were also characterized with respect to aggregation and electrophilic reactivity, and the binding modes of rhodanines and related compounds in published X-ray cocrystal structures were analyzed. The results indicate that the exocyclic, double bonded sulfur atom in rhodanines and thiohydantoins, in addition to other structural features, offers a particularly high density of interaction sites for polar interactions and hydrogen bonds. This causes a promiscuous behavior at concentrations in the "screening range" but should not be regarded as a general knockout criterion that excludes such screening hits from further development. It is suggested that special criteria for target affinity and selectivity are applied to these classes of compounds and that their exceptional and potentially valuable biomolecular binding properties are consequently exploited in a useful way. PMID:22077389

  20. The promiscuous larvae: flexibility in the establishment of symbiosis in corals

    NASA Astrophysics Data System (ADS)

    Cumbo, V. R.; Baird, A. H.; van Oppen, M. J. H.

    2013-03-01

    Coral reefs thrive in part because of the symbiotic partnership between corals and Symbiodinium. While this partnership is one of the keys to the success of coral reef ecosystems, surprisingly little is known about many aspects of coral symbiosis, in particular the establishment and development of symbiosis in host species that acquire symbionts anew in each generation. More specifically, the point at which symbiosis is established (i.e., larva vs. juvenile) remains uncertain, as does the source of free-living Symbiodinium in the environment. In addition, the capacity of host and symbiont to form novel combinations is unknown. To explore patterns of initial association between host and symbiont, larvae of two species of Acropora were exposed to sediment collected from three locations on the Great Barrier Reef. A high proportion of larvae established symbiosis shortly after contact with sediments, and Acropora larvae were promiscuous, taking up multiple types of Symbiodinium. The Symbiodinium types acquired from the sediments reflected the symbiont assemblage within a wide range of cnidarian hosts at each of the three sites, suggesting potential regional differences in the free-living Symbiodinium assemblage. Coral larvae clearly have the capacity to take up Symbiodinium prior to settlement, and sediment is a likely source. Promiscuous larvae allow species to associate with Symbiodinium appropriate for potentially novel environments that may be experienced following dispersal.

  1. Structural and Thermodymamic Basis for Enhanced DNA Binding by a Promiscuous Mutant EcoRI Endonuclease

    SciTech Connect

    Sapienza,P.; Rosenberg, J.; Jen-Jacobson, L.

    2007-01-01

    Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI*) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has S more favorable and H less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI* sites that structurally resemble the specific wild-type complex with GAATTC.

  2. Shu-Gan-Liang-Xue Decoction Simultaneously Down-regulates Expressions of Aromatase and Steroid Sulfatase in Estrogen Receptor Positive Breast Cancer Cells

    PubMed Central

    Fu, Xue-song; Li, Ping-ping

    2011-01-01

    Objective Estradiol (E2) plays an important role in the development of breast cancer. In postmenopausal women, the estrogen can be synthesized via aromatase (CYP19) pathway and steroid-sulfatase (STS) pathway in peripheral tissues, when the production in ovary has ceased. The objective of our study was to explore the effects of Shu-Gan-Liang-Xue Decoction (SGLXD) on the expressions of CYP19 and STS in estrogen receptor positive breast cancer MCF-7 and T47D cells. Methods The effects of SGLXD on the cell viability of MCF-7 and T47D were analyzed by MTT assay. By quantitative real-time RT-PCR and Western blot, we evaluated the mRNA and protein expressions of CYP19 and STS in MCF-7 and T47D cells after SGLXD treatment. Results By MTT assay, the cell viability rates of MCF-7 and T47D were significantly inhibited by SGLXD in a dose-dependent manner, the IC50 values were 40.07 mg/ml for MCF-7 cells and 25.62 mg/ml for T47D cells, respectively. As evidenced by real-time PCR and Western blot, the high concentrations of SGLXD significantly down-regulated the expressions of CYP19 and STS both in the transcript level and the protein level. Conclusion The results suggest that SGLXD is a potential dual aromatase-sulfatase inhibitor by simultaneously down-regulating the expressions of CYP19 and STS in MCF-7 and T47D cells. PMID:23467843

  3. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

    PubMed

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-03-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  4. Student Sexual Orientation, Promiscuity and Urban Acculturation as Factors That Influence Teacher Judgments about HIV[Positive] Students

    ERIC Educational Resources Information Center

    Cruce, Michael K.; Stinnett, Terry A.; Choate, Kurt T.

    2003-01-01

    Attributions toward HIV[positive] adolescents made by teacher education students who graduated from rural or urban high schools were examined. Participants read vignettes in which level of promiscuity and sexual orientation were varied, then completed a rating scale that reflected various attitudes toward HIV[positive] students. The vignette…

  5. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  6. A β-Alanine Catabolism Pathway Containing a Highly Promiscuous ω-Transaminase in the 12-Aminododecanate-Degrading Pseudomonas sp. Strain AAC

    PubMed Central

    Wilding, Matthew; Peat, Thomas S.; Newman, Janet

    2016-01-01

    ABSTRACT We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC. IMPORTANCE We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics

  7. Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis.

    PubMed

    Mariutti, Ricardo Barros; Ullah, Anwar; Araujo, Gabriela Campos; Murakami, Mario Tyago; Arni, Raghuvir Krishnaswamy

    2016-07-01

    The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium. PMID:27233609

  8. Pan Assay Interference Compounds (PAINS) and Other Promiscuous Compounds in Antifungal Research.

    PubMed

    Pouliot, Martin; Jeanmart, Stephane

    2016-01-28

    Every week, articles disclosing new antifungal leads reported as promising starting points for optimization projects are published. In many cases, the mechanism that accounts for their antifungal activity has not been fully elucidated. More significantly, the detrimental impact that could result from certain embedded chemical features has been underestimated or even overlooked. In the course of our research in the agrochemical area, we have concluded that in many cases such leads are actually nonoptimizable because they either contain what are now recognized as pan assay interference compounds (PAINS) or other promiscuous groups. This article is aimed at highlighting the pitfalls we have encountered and hopefully to steer other research groups away from them. PMID:26313340

  9. Structural Plasticity Underpins Promiscuous Binding of the Prosurvival Protein A1

    SciTech Connect

    Smits,C.; Czabotar, P.; Hinds, M.; Day, C.

    2008-01-01

    Apoptotic pathways are regulated by protein-protein interactions. Interaction of the BH3 domains of proapoptotic Bcl-2 family proteins with the hydrophobic groove of prosurvival proteins is critical. Whereas some BH3 domains bind in a promiscuous manner, others exhibit considerable selectivity and the sequence characteristics that distinguish these activities are unclear. In this study, crystal structures of complexes between the prosurvival protein A1 and the BH3 domains from Puma, Bmf, Bak, and Bid have been solved. The structure of A1 is similar to that of other prosurvival proteins, although features, such as an acidic patch in the binding groove, may allow specific therapeutic modulation of apoptosis. Significant conformational plasticity was observed in the intermolecular interactions and these differences explain some of the variation in affinity. This study, in combination with published data, suggests that interactions between conserved residues demarcate optimal binding.

  10. Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination.

    PubMed

    Cuetos, Aníbal; García-Ramos, Marina; Fischereder, Eva-Maria; Díaz-Rodríguez, Alba; Grogan, Gideon; Gotor, Vicente; Kroutil, Wolfgang; Lavandera, Iván

    2016-02-24

    Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations. PMID:26836037

  11. A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity.

    PubMed

    Sayou, Camille; Monniaux, Marie; Nanao, Max H; Moyroud, Edwige; Brockington, Samuel F; Thévenon, Emmanuel; Chahtane, Hicham; Warthmann, Norman; Melkonian, Michael; Zhang, Yong; Wong, Gane Ka-Shu; Weigel, Detlef; Parcy, François; Dumas, Renaud

    2014-02-01

    Transcription factors (TFs) are key players in evolution. Changes affecting their function can yield novel life forms but may also have deleterious effects. Consequently, gene duplication events that release one gene copy from selective pressure are thought to be the common mechanism by which TFs acquire new activities. Here, we show that LEAFY, a major regulator of flower development and cell division in land plants, underwent changes to its DNA binding specificity, even though plant genomes generally contain a single copy of the LEAFY gene. We examined how these changes occurred at the structural level and identify an intermediate LEAFY form in hornworts that appears to adopt all different specificities. This promiscuous intermediate could have smoothed the evolutionary transitions, thereby allowing LEAFY to evolve new binding specificities while remaining a single-copy gene. PMID:24436181

  12. Promiscuous Substrate Recognition in Folding and Assembly Activities of the Trigger Factor Chaperone

    SciTech Connect

    Martinez-Hackert, E.; Hendrickson, W

    2009-01-01

    Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.

  13. Promiscuous Mutations Activate the Non-Canonical NF-kB Pathway in Multiple Myeloma

    PubMed Central

    Keats, Jonathan J.; Fonseca, Rafael; Chesi, Marta; Schop, Roelandt; Baker, Angela; Chng, Wee-Joo; Van Wier, Scott; Tiedemann, Rodger; Shi, Chang-Xin; Sebag, Michael; Braggio, Esteban; Henry, Travis; Zhu, Yuan-Xiao; Fogle, Homer; Price-Troska, Tammy; Ahmann, Gregory; Mancini, Catherine; Brents, Leslie A.; Kumar, Shaji; Greipp, Philip; Dispenzieri, Angela; Bryant, Barb; Mulligan, George; Bruhn, Laurakay; Barrett, Michael; Valdez, Riccardo; Trent, Jeff; Stewart, A. Keith; Carpten, John; Bergsagel, P. Leif

    2007-01-01

    Summary Activation of NF-kB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2, and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the non-canonical NF-kB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kB pathway in the pathogenesis of multiple myeloma. PMID:17692805

  14. HUMAN PARAOXONASE-1 (PON1): GENE STRUCTURE AND EXPRESSION, PROMISCUOUS ACTIVITIES AND MULTIPLE PHYSIOLOGICAL ROLES

    PubMed Central

    Mackness, Mike; Mackness, Bharti

    2015-01-01

    Human PON1 is a HDL-associated lipolactonase capable of preventing LDL and cell membrane oxidation and is therefore considered to be atheroprotective. PON1 contributes to the antioxidative function of HDL and reductions in HDL-PON1 activity, prevalent in a wide variety of diseases with an inflammatory component, is believed to lead to dysfunctional HDL which can promote inflammation and atherosclerosis. However, PON1 is multifunctional and may contribute to other HDL functions such as in innate immunity, preventing infection by quorum sensing gram negative bacteria by destroying acyl lactone mediators of quorum sensing, and putative new roles in cancer development and the promotion of healthy ageing. In this review we explore the physiological roles of PON1 in disease development, as well as PON1 gene and protein structure, promiscuous activities and the roles of SNPs and ethnicity in determining PON1 activity. PMID:25965560

  15. The promiscuous MLL gene links chromosomal translocations to cellular differentiation and tumour tropism.

    PubMed

    Collins, Emma C; Rabbitts, Terence H

    2002-09-01

    MLL is a promiscuous gene involved in a diversity of chromosomal fusions in haematological malignancies, usually resulting from chromosomal translocations. MLL-associated chromosomal rearrangements usually occur in tumours of specific haematological lineages, suggesting a crucial role for the MLL fusion partner in determining disease phenotype (or tumour tropism). The MLL gene is homologous to Drosophila trithorax, and is likewise involved in embryo pattern formation. Common themes linking several of the MLL partners include a possible involvement in embryo patterning via Hox gene regulation and chromatin remodelling. These findings reinforce the link between developmental regulation and chromosomal translocations, and indicate the role of chromosomal translocation in activating genes capable of determining tumour phenotype in leukaemias and sarcomas. PMID:12223315

  16. Male coercion and the costs of promiscuous mating for female chimpanzees

    PubMed Central

    Muller, Martin N; Kahlenberg, Sonya M; Emery Thompson, Melissa; Wrangham, Richard W

    2007-01-01

    For reasons that are not yet clear, male aggression against females occurs frequently among primates with promiscuous mating systems. Here, we test the sexual coercion hypothesis that male aggression functions to constrain female mate choice. We use 10 years of behavioural and endocrine data from a community of wild chimpanzees (Pan troglodytes schweinfurthii) to show that sexual coercion is the probable primary function of male aggression against females. Specifically, we show that male aggression is targeted towards the most fecund females, is associated with high male mating success and is costly for the victims. Such aggression can be viewed as a counter-strategy to female attempts at paternity confusion, and a cost of multi-male mating. PMID:17264062

  17. Fidelity and Promiscuity in an Ant-Plant Mutualism: A Case Study of Triplaris and Pseudomyrmex

    PubMed Central

    Sanchez, Adriana

    2015-01-01

    The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism but no molecular studies have examined this association to date. In this study, the interspecific relationships of Triplaris were reconstructed using five molecular markers (two chloroplast and three nuclear), and the relationships of the associated Pseudomyrmex using two molecular regions (one mitochondrial and one nuclear). A data set including all known collections of plant hosts and resident ants was also compiled. The pattern of distribution of both organisms reveals that there are varying degrees of host specificity; most ants show broader host usage (promiscuous) but one species (P. dendroicus) is faithful to a single species of Triplaris. In most ant-plant interactions, host usage is not specific at the species level and preferences may result from geographical or ecological sorting. The specificity of P. dendroicus could be based on chemical recognition of the host they were raised on. PMID:26630384

  18. Synthesis and Structure–Activity Relationship Studies of Derivatives of the Dual Aromatase–Sulfatase Inhibitor 4-{[(4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino]methyl}phenyl sulfamate

    PubMed Central

    Woo, L W Lawrence; Wood, Paul M; Bubert, Christian; Thomas, Mark P; Purohit, Atul; Potter, Barry V L

    2013-01-01

    4-{[(4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino]methyl}phenyl sulfamate and its ortho-halogenated (F, Cl, Br) derivatives are first-generation dual aromatase and sulfatase inhibitors (DASIs). Structure–activity relationship studies were performed on these compounds, and various modifications were made to their structures involving relocation of the halogen atom, introduction of more halogen atoms, replacement of the halogen with another group, replacement of the methylene linker with a difluoromethylene linker, replacement of the para-cyanophenyl ring with other ring structures, and replacement of the triazolyl group with an imidazolyl group. The most potent in vitro DASI discovered is an imidazole derivative with IC50 values against aromatase and steroid sulfatase in a JEG-3 cell preparation of 0.2 and 2.5 nm, respectively. The parent phenol of this compound inhibits aromatase with an IC50 value of 0.028 nm in the same assay. PMID:23495205

  19. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

    PubMed

    Eudes, Aymerick; Pereira, Jose H; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E K; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D; Loqué, Dominique

    2016-03-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  20. Males and Females Gain Differentially from Sociality in a Promiscuous Fruit Bat Cynopterus sphinx

    PubMed Central

    Garg, Kritika M.; Chattopadhyay, Balaji; Swami Doss, D. P.; Kumar, A. K. Vinoth; Kandula, Sripathi; Ramakrishnan, Uma

    2015-01-01

    Sociality emerges when the benefits of group living outweigh its costs. While both males and females are capable of strong social ties, the evolutionary drivers for sociality and the benefits accrued maybe different for each sex. In this study, we investigate the differential reproductive success benefits of group membership that males and females might obtain in the promiscuous fruit bat Cynopterus sphinx. Individuals of this species live in flexible social groups called colonies. These colonies are labile and there is high turnover of individuals. However, colony males sire more offspring within the colony suggesting that being part of a colony may result in reproductive benefits for males. This also raises the possibility that long-term loyalty towards the colony may confer additional advantage in terms of higher reproductive success. We used ten seasons of genetic parentage data to estimate reproductive success and relatedness of individuals in the colony. We used recapture data to identify long and short-term residents in the colony as well as to obtain rates of recapture for males and females. Our results reveal that males have a significantly higher chance of becoming long-term residents (than females), and these long-term resident males gain twice the reproductive success compared to short-term resident males. We also observed that long-term resident females are related to each other and also achieve higher reproductive success than short-term resident females. In contrast, long-term resident males do not differ from short-term resident males in their levels of relatedness. Our results re-iterate the benefits of sociality even in species that are promiscuous and socially labile and possible benefits of maintaining a colony. PMID:25794185

  1. Males and females gain differentially from sociality in a promiscuous fruit bat Cynopterus sphinx.

    PubMed

    Garg, Kritika M; Chattopadhyay, Balaji; Swami Doss, D P; Kumar, A K Vinoth; Kandula, Sripathi; Ramakrishnan, Uma

    2015-01-01

    Sociality emerges when the benefits of group living outweigh its costs. While both males and females are capable of strong social ties, the evolutionary drivers for sociality and the benefits accrued maybe different for each sex. In this study, we investigate the differential reproductive success benefits of group membership that males and females might obtain in the promiscuous fruit bat Cynopterus sphinx. Individuals of this species live in flexible social groups called colonies. These colonies are labile and there is high turnover of individuals. However, colony males sire more offspring within the colony suggesting that being part of a colony may result in reproductive benefits for males. This also raises the possibility that long-term loyalty towards the colony may confer additional advantage in terms of higher reproductive success. We used ten seasons of genetic parentage data to estimate reproductive success and relatedness of individuals in the colony. We used recapture data to identify long and short-term residents in the colony as well as to obtain rates of recapture for males and females. Our results reveal that males have a significantly higher chance of becoming long-term residents (than females), and these long-term resident males gain twice the reproductive success compared to short-term resident males. We also observed that long-term resident females are related to each other and also achieve higher reproductive success than short-term resident females. In contrast, long-term resident males do not differ from short-term resident males in their levels of relatedness. Our results re-iterate the benefits of sociality even in species that are promiscuous and socially labile and possible benefits of maintaining a colony. PMID:25794185

  2. Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme.

    PubMed

    Zou, Taisong; Risso, Valeria A; Gavira, Jose A; Sanchez-Ruiz, Jose M; Ozkan, S Banu

    2015-01-01

    β-Lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity. PMID:25312912

  3. Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels*

    PubMed Central

    Redaelli, Elisa; Cassulini, Rita Restano; Silva, Deyanira Fuentes; Clement, Herlinda; Schiavon, Emanuele; Zamudio, Fernando Z.; Odell, George; Arcangeli, Annarosa; Clare, Jeffrey J.; Alagón, Alejandro; de la Vega, Ricardo C. Rodríguez; Possani, Lourival D.; Wanke, Enzo

    2010-01-01

    Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their “canonical” gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-à-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs. PMID:19955179

  4. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    PubMed Central

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E.K.; Lee, Taek Soon; Adams, Paul D.; Keasling, Jay D.; Loqué, Dominique

    2016-01-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  5. Promiscuous catalysis of asymmetric Michael-type additions of linear aldehydes to β-nitrostyrene by the proline-based enzyme 4-oxalocrotonate tautomerase.

    PubMed

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

    2013-01-21

    Exploiting catalytic promiscuity: The proline-based enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes asymmetric Michael-type additions of linear aldehydes--ranging from acetaldehyde to octanal--to trans-β-nitrostyrene in aqueous solvent. The presence of 1.4 mol% of 4-OT effected formation of the anticipated γ-nitroaldehydes in fair to good yields with dr values of up to 93:7 and ee values of up to 81 %. PMID:23303727

  6. PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

    PubMed Central

    2015-01-01

    Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter. PMID:25634295

  7. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  8. Coexistence of unlimited bipartite and genuine multipartite entanglement: Promiscuous quantum correlations arising from discrete to continuous-variable systems

    SciTech Connect

    Adesso, Gerardo; Ericsson, Marie; Illuminati, Fabrizio

    2007-08-15

    Quantum mechanics imposes 'monogamy' constraints on the sharing of entanglement. We show that, despite these limitations, entanglement can be fully 'promiscuous', i.e., simultaneously present in unlimited two-body and many-body forms in states living in an infinite-dimensional Hilbert space. Monogamy just bounds the divergence rate of the various entanglement contributions. This is demonstrated in simple families of N-mode (N{>=}4) Gaussian states of light fields or atomic ensembles, which therefore enable infinitely more freedom in the distribution of information, as opposed to systems of individual qubits. Such a finding is of importance for the quantification, understanding, and potential exploitation of shared quantum correlations in continuous variable systems. We discuss how promiscuity gradually arises when considering simple families of discrete variable states, with increasing Hilbert space dimension towards the continuous variable limit. Such models are somehow analogous to Gaussian states with asymptotically diverging, but finite, squeezing. In this respect, we find that non-Gaussian states (which in general are more entangled than Gaussian states) exhibit also the interesting feature that their entanglement is more shareable: in the non-Gaussian multipartite arena, unlimited promiscuity can be already achieved among three entangled parties, while this is impossible for Gaussian, even infinitely squeezed states.

  9. Coexistence of unlimited bipartite and genuine multipartite entanglement: Promiscuous quantum correlations arising from discrete to continuous-variable systems

    NASA Astrophysics Data System (ADS)

    Adesso, Gerardo; Ericsson, Marie; Illuminati, Fabrizio

    2007-08-01

    Quantum mechanics imposes “monogamy” constraints on the sharing of entanglement. We show that, despite these limitations, entanglement can be fully “promiscuous,” i.e., simultaneously present in unlimited two-body and many-body forms in states living in an infinite-dimensional Hilbert space. Monogamy just bounds the divergence rate of the various entanglement contributions. This is demonstrated in simple families of N -mode (N⩾4) Gaussian states of light fields or atomic ensembles, which therefore enable infinitely more freedom in the distribution of information, as opposed to systems of individual qubits. Such a finding is of importance for the quantification, understanding, and potential exploitation of shared quantum correlations in continuous variable systems. We discuss how promiscuity gradually arises when considering simple families of discrete variable states, with increasing Hilbert space dimension towards the continuous variable limit. Such models are somehow analogous to Gaussian states with asymptotically diverging, but finite, squeezing. In this respect, we find that non-Gaussian states (which in general are more entangled than Gaussian states) exhibit also the interesting feature that their entanglement is more shareable: in the non-Gaussian multipartite arena, unlimited promiscuity can be already achieved among three entangled parties, while this is impossible for Gaussian, even infinitely squeezed states.

  10. Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations

    SciTech Connect

    Singla, Nikhil; Goldgur, Yehuda; Xu, Kai; Paavilainen, Sari; Nikolov, Dimitar B.; Himanen, Juha P.

    2010-09-08

    Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3 {angstrom} resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.

  11. Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: Evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes

    PubMed Central

    Wei, Wei; Miller, Rebecca L.; Leary, Julie A.

    2013-01-01

    Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis, however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration and solid phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated. PMID:23659730

  12. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

    DOE PAGESBeta

    Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S.; Ben-Tal, Nir; Dwyer, Daniel J.; Gophna, Uri; et al

    2016-01-28

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

  13. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

    PubMed Central

    Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S.; Ben-Tal, Nir; Dwyer, Daniel J.; Gophna, Uri; Ruppin, Eytan

    2016-01-01

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly. PMID:26821166

  14. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli.

    PubMed

    Oberhardt, Matthew A; Zarecki, Raphy; Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S; Ben-Tal, Nir; Dwyer, Daniel J; Gophna, Uri; Ruppin, Eytan

    2016-01-01

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly. PMID:26821166

  15. Disorder, Promiscuous Interactions, and Stochasticity Regulate State Switching in the Unstable Prostate.

    PubMed

    Kulkarni, Prakash; Getzenberg, Robert H

    2016-10-01

    A causal link between benign prostatic hyperplasia (BPH) and prostate cancer has long been suspected but not widely accepted. A new model is proposed that supports such a connection. In contrast to the prevailing wisdom, our model, that draws on dynamical systems theory, suggests that in response to stress, epithelial cells in the unstable gland can give rise to both types of diseases via a phenotypic switching mechanism. The central idea is that phenotypic switching is a stochastic process which exploits the plasticity of the epithelial cell. It is driven by 'noise' contributed by the conformational dynamics of proteins that are intrinsically disordered. In a system that is noisy when stressed, disorder promotes promiscuity, unmasks latent information, and rewires the network to cause phenotypic switching. Cells with newly acquired phenotypes can transcend the traditional zonal boundaries to give rise to BPH or prostate cancer depending on the microenvironment. Establishing causality between the two diseases may provide us with an opportunity to better understand their etiology and guide prevention and treatment strategies. J. Cell. Biochem. 117: 2235-2240, 2016. © 2016 Wiley Periodicals, Inc. PMID:27152744

  16. NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling

    SciTech Connect

    Mace, P.D.; Robinson, H.; Wallez, Y.; Dobaczewska, M. K.; Lee, J. J.; Pasquale, E. B.; Riedl, S. J.

    2011-12-01

    Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas. BCAR3 adopts the Cdc25-homology fold of Ras GTPase exchange factors, but it has a 'closed' conformation incapable of enzymatic activity. The structure of the NSP3-p130Cas complex reveals that this closed conformation is instrumental for interaction of NSP proteins with a focal adhesion-targeting domain present in Cas proteins. This enzyme-to-adaptor conversion enables high-affinity, yet promiscuous, interactions between NSP and Cas proteins and represents an unprecedented mechanistic paradigm linking cellular signaling networks.

  17. Probing the Catalytic Promiscuity of a Regio- and Stereospecific C-Glycosyltransferase from Mangifera indica.

    PubMed

    Chen, Dawei; Chen, Ridao; Wang, Ruishan; Li, Jianhua; Xie, Kebo; Bian, Chuancai; Sun, Lili; Zhang, Xiaolin; Liu, Jimei; Yang, Lin; Ye, Fei; Yu, Xiaoming; Dai, Jungui

    2015-10-19

    The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery. PMID:26331569

  18. The Under-Appreciated Promiscuity of the Epidermal Growth Factor Receptor Family.

    PubMed

    Kennedy, Sean P; Hastings, Jordan F; Han, Jeremy Z R; Croucher, David R

    2016-01-01

    Each member of the epidermal growth factor receptor (EGFR) family plays a key role in normal development, homeostasis, and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3, and ERBB4) function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signaling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance toward targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies. PMID:27597943

  19. Synthesis, characterization, hydrolase and catecholase activity of a dinuclear iron(III) complex: Catalytic promiscuity.

    PubMed

    Camargo, Tiago P; Maia, Fernanda F; Chaves, Cláudia; de Souza, Bernardo; Bortoluzzi, Adailton J; Castilho, Nathalia; Bortolotto, Tiago; Terenzi, Hernán; Castellano, Eduardo E; Haase, Wolfgang; Tomkowicz, Zbigniew; Peralta, Rosely A; Neves, Ademir

    2015-05-01

    Herein, we report the synthesis and characterization of the new di-iron(III) complex [(bbpmp)(H2O)(Cl)Fe(III)(μ-Ophenoxo)Fe(III)(H2O)Cl)]Cl (1), with the symmetrical ligand 2,6-bis{[(2-hydroxybenzyl)(pyridin-2-yl)methylamino]methyl}-4-methylphenol (H3bbpmp). Complexes 2 with the unsymmetrical ligand H2bpbpmp - {2-[[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl]-6-bis(pyridylmethyl) aminomethyl}-4-methylphenol and 3 with the ligand L(1)=4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam were included for comparison purposes. Complex 1 was characterized through elemental analysis, X-ray crystallography, magnetochemistry, electronic spectroscopy, electrochemistry, mass spectrometry and potentiometric titration. The magnetic data show a very weak antiferromagnetic coupling between the two iron centers of the dinuclear complex 1 (J=-0.29cm(-1)). Due to the presence of labile coordination sites in both iron centers the hydrolysis of both the diester model substrate 2,4-BDNPP and DNA was studied in detail. Complex 1 was also able to catalyze the oxidation of the substrate 3,5-di-tert-butylcatechol (3,5-DTBC) to give the corresponding quinone, and thus it can be considered as a catalytically promiscuous system. PMID:25792035

  20. The Under-Appreciated Promiscuity of the Epidermal Growth Factor Receptor Family

    PubMed Central

    Kennedy, Sean P.; Hastings, Jordan F.; Han, Jeremy Z. R.; Croucher, David R.

    2016-01-01

    Each member of the epidermal growth factor receptor (EGFR) family plays a key role in normal development, homeostasis, and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3, and ERBB4) function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signaling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance toward targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies. PMID:27597943

  1. Promiscuous and specific bacterial symbiont acquisition in the amoeboid genus Nuclearia (Opisthokonta).

    PubMed

    Dirren, Sebastian; Posch, Thomas

    2016-08-01

    We isolated 17 strains of the amoeboid genus Nuclearia (Opisthokonta) from five Swiss lakes. Eight of these nucleariid isolates were associated with bacterial endosymbionts and/or ectosymbionts. Amoebae were characterized morphologically and by their 18S rRNA genes. Phylogeny based on molecular data resulted in four established monophyletic branches and two new clusters. A heterogeneous picture emerged by highlighting nucleariids with associated bacteria. Apart from one cluster which consisted of only isolates with and three groups of amoebae without symbionts, we also found mixed clusters. The picture got even more 'blurred' by regarding the phylogeny of symbiotic bacteria. Although seven different bacterial strains could be identified, it seems that we still are only scratching the surface of symbionts' diversity. Furthermore, types of symbioses might be different depending on host species. Strains of Nuclearia thermophila harboured the same endosymbiont even when isolated from different lakes. This pointed to a specific and obligate interaction. However, two isolates of N. delicatula were associated with different endosymbiotic bacteria. Here the symbiont acquisition seemed to be rather promiscuous. This behaviour regarding symbiotic associations is especially remarkable considering the phylogenetic position of these basal opisthokonts. PMID:27199347

  2. Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

    PubMed Central

    Clawson, Michael L.; Benson, David R.

    1999-01-01

    Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N2-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia. PMID:10508084

  3. Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis.

    PubMed

    Mafu, Sibongile; Jia, Meirong; Zi, Jiachen; Morrone, Dana; Wu, Yisheng; Xu, Meimei; Hillwig, Matthew L; Peters, Reuben J

    2016-03-01

    The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis. PMID:26884192

  4. Promiscuous Gene Expression in the Thymus: A Matter of Epigenetics, miRNA, and More?

    PubMed Central

    Ucar, Olga; Rattay, Kristin

    2015-01-01

    The induction of central tolerance in the course of T cell development crucially depends on promiscuous gene expression (pGE) in medullary thymic epithelial cells (mTECs). mTECs express a genome-wide variety of tissue-restricted antigens (TRAs), preventing the escape of autoreactive T cells to the periphery, and the development of severe autoimmunity. Most of our knowledge of how pGE is controlled comes from studies on the autoimmune regulator (Aire). Aire activates the expression of a large subset of TRAs by interacting with the general transcriptional machinery and promoting transcript elongation. However, further factors regulating Aire-independent TRAs must be at play. Recent studies demonstrated that pGE in general and the function of Aire in particular are controlled by epigenetic and post-transcriptional mechanisms. This mini-review summarizes current knowledge of the regulation of pGE by miRNA and epigenetic regulatory mechanisms such as DNA methylation, histone modifications, and chromosomal topology. PMID:25784915

  5. AIDS and the stigma of sexual promiscuity: Thai nurses' risk perceptions of occupational exposure to HIV.

    PubMed

    Chan, Kit Yee; Rungpueng, Arattha; Reidpath, Daniel D

    2009-05-01

    This paper examines the culturally shaped meanings of AIDS and perceptions of accidental occupational exposure to HIV among a group of twenty nurses in Bangkok, Thailand. The findings are based on data collected as a part of a larger mixed-methods study that examined how perceptions of risk behaviours (including sexual promiscuity) shape health workers' perceptions of patients living with HIV/AIDS. Nurses' narratives revealed that despite acknowledgement of the low probability of occupational exposure to HIV, the fear of HIV infection remained and was largely driven by the enormity of the anticipated social (rather than the health) consequences of being HIV-positive. The perceived certainty of social ostracism was reinforced by participants' observations of the social rejection experienced by people living with HIV/AIDS both within and outside clinical settings. For female nurses, the dominant social perception that women living with HIV/AIDS were violators of gender norms, and thus 'guilty' victims, was an issue central to their self-identities. Ways of improving care for people living with HIV in the light of the nurses' concerns and future research are discussed. PMID:19263260

  6. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    PubMed

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies. PMID:26187411

  7. Epitope-specificity of recombinant antibodies reveals promiscuous peptide-binding properties

    PubMed Central

    Olsson, Niclas; Wallin, Stefan; James, Peter; Borrebaeck, Carl A K; Wingren, Christer

    2012-01-01

    Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large-scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide-binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low-affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide-binding specificity appeared to be executed through the use of 2–3 semi-conserved anchor residues in the C-terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long-term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide-binding proteins in general, in various biotechnical and medical applications. PMID:23034898

  8. The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing

    PubMed Central

    Abe, Ken-ichiro; Yamamoto, Ryoma; Franke, Vedran; Cao, Minjun; Suzuki, Yutaka; Suzuki, Masataka G; Vlahovicek, Kristian; Svoboda, Petr; Schultz, Richard M; Aoki, Fugaku

    2015-01-01

    Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos. PMID:25896510

  9. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

    PubMed Central

    Ye, Zhixia; Musiol, Ewa M; Weber, Tilmann; Williams, Gavin J

    2014-01-01

    SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues. PMID:24726832

  10. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.

    PubMed

    Roux, Kyle J; Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-03-19

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  11. The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs

    PubMed Central

    Norvaisas, Povilas; Chan, Diana; Yokoi, Kenji; Dave, Bhuvanesh

    2016-01-01

    NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future. PMID:25714784

  12. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  13. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    PubMed

    Ueda, Shuji; Blee, Alexandra M; Macway, Katherine G; Renner, Derrick J; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  14. Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

    PubMed

    Peng, Jianhe; Alam, Sarfaraz; Radhakrishnan, Karthikeyan; Mariappan, Malaiyalam; Rudolph, Markus Georg; May, Caroline; Dierks, Thomas; von Figura, Kurt; Schmidt, Bernhard

    2015-09-01

    C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor. PMID:26077311

  15. The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions

    PubMed Central

    Piedrafita, Gabriel; Keller, Markus A; Ralser, Markus

    2015-01-01

    Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease—age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network. PMID:26378592

  16. Enzyme replacement therapy for mucopolysaccharidosis VI: evaluation of long-term pulmonary function in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase.

    PubMed

    Harmatz, Paul; Yu, Zi-Fan; Giugliani, Roberto; Schwartz, Ida Vanessa D; Guffon, Nathalie; Teles, Elisa Leão; Miranda, M Clara Sá; Wraith, J Edmond; Beck, Michael; Arash, Laila; Scarpa, Maurizio; Ketteridge, David; Hopwood, John J; Plecko, Barbara; Steiner, Robert; Whitley, Chester B; Kaplan, Paige; Swiedler, Stuart J; Hardy, Karen; Berger, Kenneth I; Decker, Celeste

    2010-02-01

    Pulmonary function is impaired in untreated mucopolysaccharidosis type VI (MPS VI). Pulmonary function was studied in patients during long-term enzyme replacement therapy (ERT) with recombinant human arylsulfatase B (rhASB; rhN-acetylgalactosamine 4-sulfatase). Pulmonary function tests prior to and for up to 240 weeks of weekly infusions of rhASB at 1 mg/kg were completed in 56 patients during Phase 1/2, Phase 2, Phase 3 and Phase 3 Extension trials of rhASB and the Survey Study. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and, in a subset of patients, maximum voluntary ventilation (MVV), were analyzed as absolute volume in liters. FEV1 and FVC showed little change from baseline during the first 24 weeks of ERT, but after 96 weeks, these parameters increased over baseline by 11% and 17%, respectively. This positive trend compared with baseline continued beyond 96 weeks of treatment. Improvements from baseline in pulmonary function occurred along with gains in height in the younger group (5.5% change) and in the older patient group (2.4% change) at 96 weeks. Changes in MVV occurred earlier within 24 weeks of treatment to approximately 15% over baseline. Model results based on data from all trials showed significant improvements in the rate of change in pulmonary function during 96 weeks on ERT, whereas little or no improvement was observed for the same time period prior to ERT. Thus, analysis of mean percent change data and longitudinal modeling both indicate that long-term ERT resulted in improvement in pulmonary function in MPS VI patients. PMID:20140523

  17. 24-hydroxycholesterol sulfation by human cytosolic sulfotransferases: formation of monosulfates and disulfates, molecular modeling, sulfatase sensitivity, and inhibition of liver x receptor activation.

    PubMed

    Cook, Ian T; Duniec-Dmuchowski, Zofia; Kocarek, Thomas A; Runge-Morris, Melissa; Falany, Charles N

    2009-10-01

    24-Hydroxycholesterol (24-OHChol) is a major cholesterol metabolite and the form in which cholesterol is secreted from the brain. 24-OHChol is transported by apolipoprotein E to the liver and converted into bile acids or excreted. In both brain and liver, 24-OHChol is a liver X receptor (LXR) agonist and has an important role in cholesterol homeostasis. 24-OHChol sulfation was examined to understand its role in 24-OHChol metabolism and its effect on LXR activation. 24-OHChol was conjugated by three isoforms of human cytosolic sulfotransferase (SULT). SULT2A1 and SULT1E1 sulfated both the 3- and 24-hydroxyls to form the 24-OHChol-3, 24-disulfate. SULT2B1b formed only 24-OHChol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate was hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate was resistant. At physiological 24-OHChol concentrations, SULT2A1 formed the 3-monosulfate and the 3, 24-disulfate as a result of a high affinity for sulfation of the 3-OH in 24-OHChol-24-sulfate. Molecular docking simulations indicate that 24-OHChol-24-sulfate binds in an active configuration in the SULT2A1 substrate binding site with high affinity only when the SULT2A1 homodimer structure was used. 24-OHChol is an LXR activator. In contrast, the 24-OHChol monosulfates were not LXR agonists in a fluorescence resonance energy transfer coactivator recruitment assay. However, both the 24-OHChol-3-sulfate and 24-sulfate were antagonists of LXR activation by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trif-luoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) with an IC(50) of 0.15 and 0.31 muM, respectively. Inhibition of LXR activation by the 24-OHChol monosulfates at low nanomolar concentrations indicates that sulfation has a role in LXR regulation by oxysterols. PMID:19589875

  18. The Nuclear Hormone Receptor NHR-40 Acts Downstream of the Sulfatase EUD-1 as Part of a Developmental Plasticity Switch in Pristionchus.

    PubMed

    Kieninger, Manuela R; Ivers, Nicholas A; Rödelsperger, Christian; Markov, Gabriel V; Sommer, Ralf J; Ragsdale, Erik J

    2016-08-22

    Developmental plasticity, the ability of one genotype to produce distinct phenotypes in different environments, has been suggested to facilitate phenotypic diversification, and several examples in plants and animals support its macroevolutionary potential [1-8]. However, little is known about associated molecular mechanisms, because environmental effects on development are difficult to study by laboratory approaches. One promising system is the mouth dimorphism of the nematode Pristionchus pacificus [9-12]. Following an irreversible decision in larval development, these nematodes form moveable teeth that occur in either of two discrete morphs. The "eurystomatous" (Eu) form has a wide mouth and two teeth, allowing predatory feeding on other nematodes. In contrast, the alternative ("stenostomatous"; St) form has diminutive mouthparts that largely constrain its diet to microbes. The sulfatase EUD-1 was previously discovered to execute a polyphenism switch based on dosage of functional alleles [13] and confirmed a prediction of evolutionary theory about how developmental switches control plasticity [1, 3]. However, the genetic context of this single gene, and hence the molecular complexity of switch mechanisms, was previously unknown. Here we use a suppressor screen to identify factors downstream of eud-1 in mouth-form regulation. We isolated three dominant, X-linked mutants in the nuclear hormone receptor gene nhr-40 that are haploinsufficient. Both eud-1 nhr-40 double and nhr-40 single mutants are all Eu, whereas transgenic overexpression of nhr-40 does not restore the wild-type phenotype but instead results in nearly all-St lines. Thus, NHR-40 is part of a developmental switch, suggesting that switch mechanisms controlling plasticity consist of multi-component hormonal signaling systems. PMID:27451902

  19. Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF

    PubMed Central

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

    2016-01-01

    Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events. PMID:27078017

  20. Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: toward mutation mapping of the iduronate-2-sulfatase gene.

    PubMed Central

    Jonsson, J J; Aronovich, E L; Braun, S E; Whitley, C B

    1995-01-01

    Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C-->T, severe phenotype); P358R (1073C-->G, severe); R468W (1402C-->T, mild); P469H (1406C-->A, mild); and Y523C (1568A-->G, mild). Non-sense mutations were identified in two patients: R172X (514C-->T, severe) and Q389X (1165C-->T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (> 95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395 C-->G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented. Images Figure 2 PMID:7887413

  1. Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients.

    PubMed Central

    Litjens, T.; Brooks, D. A.; Peters, C.; Gibson, G. J.; Hopwood, J. J.

    1996-01-01

    Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients. PMID:8651289

  2. Immunologic Hierarchy, Class II MHC Promiscuity, and Epitope Spreading of a Melanoma Helper Peptide Vaccine

    PubMed Central

    Hu, Yinin; Petroni, Gina R.; Olson, Walter C.; Czarkowski, Andrea; Smolkin, Mark E.; Grosh, William W.; Chianese-Bullock, Kimberly A.; Slingluff, Craig L.

    2014-01-01

    Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4+ T-cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides, and identify helper peptide-mediated augmentation of specific CD8+ T-cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1 and cancer-testis antigens from the MAGE family. CD4+ and CD8+ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELI spot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4+ T cell responses to individual epitopes were detected in the SIN of 63% (22/35) and in the peripheral blood of 38% (14/37) of participants for an overall response rate of 65% (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49%) and tyrosinase 386-406 (32%). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8+ T-cell responses against class I-restricted peptides were observed in 45% (5/11) of evaluable participants. The 6MHP vaccine induces both CD4+ and CD8 + T cell responses against melanoma antigens. CD4+ T-cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8+ T-cell responses suggests epitope spreading and systemic activity mediated at the tumor site. PMID:24756419

  3. Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

    PubMed Central

    Soo, Valerie W. C.; Hanson-Manful, Paulina; Patrick, Wayne M.

    2011-01-01

    Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004–17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations. PMID:21173244

  4. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

    PubMed

    Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

    2016-02-25

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process. PMID:26863189

  5. A promiscuous recognition mechanism between GPR17 and SDF-1: Molecular insights.

    PubMed

    Parravicini, Chiara; Daniele, Simona; Palazzolo, Luca; Trincavelli, Maria Letizia; Martini, Claudia; Zaratin, Paola; Primi, Roberto; Coppolino, Giusy; Gianazza, Elisabetta; Abbracchio, Maria P; Eberini, Ivano

    2016-06-01

    Recent data and publications suggest a promiscuous behaviour for GPR17, a class-A GPCR operated by different classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. This observation, together with the ability of several class-A GPCRs to form homo- and hetero-dimers, is likely to unveil new pathophysiological roles and novel emerging pharmacological properties for some of these GPCRs, including GPR17. This receptor shares structural, phylogenetic and functional properties with some chemokine receptors, CXCRs. Both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCR ligands have been demonstrated to have a role in orchestrating inflammatory responses and oligodendrocyte precursor cell differentiation to myelinating cells in acute and chronic diseases of the central nervous system. Here, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary oligodendrocyte precursor cells, we demonstrate that GPR17 can be activated by SDF-1, a ligand of chemokine receptors CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to different classes of GPCR ligands suggests that this receptor modifies its function depending on the extracellular mileu changes occurring under specific pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an inflammatory/immune component. PMID:26971834

  6. Identification of promiscuous HPV16-derived T helper cell epitopes for therapeutic HPV vaccine design.

    PubMed

    Grabowska, Agnieszka K; Kaufmann, Andreas M; Riemer, Angelika B

    2015-01-01

    Cervical carcinoma and several other human papillomavirus (HPV)-induced malignancies are a global public health problem, thus novel treatment modalities are urgently needed. Immunotherapy is an attractive option for treatment of HPV infection and HPV-mediated premalignant and malignant lesions. However, previous approaches--focusing on the induction of cytotoxic CD8+ T cells (CTLs)--have as yet not yielded clinical successes. Since CD4+ T cells have been shown to be crucial for the induction and maintenance of CTL responses, and more recently to be also important for direct anti-tumor immunity, human leukocyte antigen (HLA) class II-restricted epitopes are intensively investigated to improve the efficacy of peptide-based HPV immunotherapy. We here present an approach to identify promiscuous HPV16-derived CD4+ T helper epitopes, which are capable of inducing T cell immunity in a large proportion of the population. To this end, we combined HLA class II epitope prediction servers with in vitro immunological evaluation to identify HPV16 E2-, E5-, E6-, and E7-derived CD4+ T cell epitopes. Candidate selected HPV16-derived epitopes were found to be restricted by up to nine HLA-DR molecules. Furthermore, they were found to induce frequent and robust HPV16 peptide-specific Th1 responses in healthy donors, as monitored by interferon (IFN)-γ ELISPOT and cytokine secretion assays. Moreover, these selected peptides also induced specific IFN-γ T cell responses in blood from HPV16+ CIN2/3 and cervical carcinoma patients. We thus conclude that the identified T helper epitopes are valuable candidates for the development of a comprehensive therapeutic HPV vaccine. PMID:24824905

  7. Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

    PubMed Central

    Nascimento, Eduardo J. M.; Mailliard, Robbie B.; Khan, Asif M.; Sidney, John; Sette, Alessandro; Guzman, Nicole; Paulaitis, Michael; de Melo, Andréa Barbosa; Cordeiro, Marli T.; Gil, Laura V. G.; Lemonnier, Françoir; Rinaldo, Charles; August, J. Thomas; Marques, Ernesto T. A.

    2013-01-01

    Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. PMID:24130917

  8. Biophysical basis of the promiscuous binding of B-cell lymphoma protein 2 apoptotic repressor to BH3 ligands.

    PubMed

    Bhat, Vikas; Olenick, Max B; Schuchardt, Brett J; Mikles, David C; McDonald, Caleb B; Farooq, Amjad

    2013-10-01

    B-cell lymphoma protein 2 (Bcl2) apoptotic repressor carries out its function by virtue of its ability to bind to BH3 domains of various pro-apoptotic regulators in a highly promiscuous manner. Herein, we investigate the biophysical basis of such promiscuity of Bcl2 toward its cognate BH3 ligands. Our data show that although the BH3 ligands harboring the LXXXAD motif bind to Bcl2 with submicromolar affinity, those with the LXXX[G/S]D motif afford weak interactions. This implies that the replacement of alanine at the fourth position (A + 4)-relative to the N-terminal leucine (L0) within the LXXXAD motif-to glycine/serine results in the loss of free energy of binding. Consistent with this notion, the A + 4 residue within the BH3 ligands harboring the LXXXAD motif engages in key intermolecular van der Waals contacts with A149 lining the ligand binding groove within Bcl2, whereas A + 4G/S substitution results in the disruption of such favorable binding interactions. Of particular interest is the observation that although increasing ionic strength has little or negligible effect on the binding of high-affinity BH3 ligands harboring the LXXXAD motif, the binding of those with the LXXX[G/S]D motif in general experiences a varying degree of enhancement. This salient observation is indicative of the fact that hydrophobic forces not only play a dominant but also a universal role in driving the Bcl2-BH3 interactions. Taken together, our study sheds light on the molecular basis of the factors governing the promiscuous binding of Bcl2 to pro-apoptotic regulators and thus bears important consequences on the development of rational therapeutic approaches. PMID:23996493

  9. Promiscuous Girls, Good Wives, and Cheating Husbands: Gender Inequality, Transitions to Marriage, and Infidelity in Southeastern Nigeria

    PubMed Central

    Smith, Daniel Jordan

    2013-01-01

    The transition from premarital sexual relationships and courtship to marriage and parenthood in southeastern Nigeria involves particularly dramatic adjustments for young women who have absorbed changing ideas about sexuality, marriage, and gender equality, and who have had active premarital sexual lives. In the eyes of society, these women must transform from being promiscuous girls to good wives. This paper examines these adjustments and, specifically, how young married women’s lives are affected by the reality of male infidelity and a persistent gendered double standard regarding the acceptability of extramarital sex. PMID:24259752

  10. Versatility or Promiscuity: The Estrogen Receptors, Control of Ligand Selectivity and an Update on Subtype Selective Ligands

    PubMed Central

    Ng, Hui Wen; Perkins, Roger; Tong, Weida; Hong, Huixiao

    2014-01-01

    The estrogen receptors (ERs) are a group of versatile receptors. They regulate an enormity of processes starting in early life and continuing through sexual reproduction, development, and end of life. This review provides a background and structural perspective for the ERs as part of the nuclear receptor superfamily and discusses the ER versatility and promiscuity. The wide repertoire of ER actions is mediated mostly through ligand-activated transcription factors and many DNA response elements in most tissues and organs. Their versatility, however, comes with the drawback of promiscuous interactions with structurally diverse exogenous chemicals with potential for a wide range of adverse health outcomes. Even when interacting with endogenous hormones, ER actions can have adverse effects in disease progression. Finally, how nature controls ER specificity and how the subtle differences in receptor subtypes are exploited in pharmaceutical design to achieve binding specificity and subtype selectivity for desired biological response are discussed. The intent of this review is to complement the large body of literature with emphasis on most recent developments in selective ER ligands. PMID:25162709

  11. In Silico Prediction of Inhibition of Promiscuous Breast Cancer Resistance Protein (BCRP/ABCG2)

    PubMed Central

    Ding, Yi-Lung; Shih, Yu-Hsuan; Tsai, Fu-Yuan; Leong, Max K.

    2014-01-01

    Background Breast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart. Methodology/Principal Findings An in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n = 22, r2 = 0.82,  = 0.73, RMSE  =  0.40, s = 0.24), test set (n = 97, q2 = 0.75–0.89, RMSE  = 0.31, s = 0.21), and outlier set (n = 16, q2 = 0.72–0.91, RMSE  =  0.29, s = 0.17). When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity. Conclusions/Significance It was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug–drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance. PMID:24614353

  12. Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

    PubMed

    James, M R; Skaar, T C; Lee, R Y; MacPherson, A; Zwiebel, J A; Ahluwalia, B S; Ampy, F; Clarke, R

    2001-04-01

    Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable

  13. Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of chemodiversity in Nature (2010 JGI User Meeting)

    ScienceCinema

    Noel, Joseph

    2011-04-25

    Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting

  14. Parents' Divorce Is More Strongly Related to the Self-Perceived Promiscuity and Drinking Behavior of Male than of Female College Students

    ERIC Educational Resources Information Center

    Stringfellow, Erica L.; McAndrew, Francis T.

    2010-01-01

    A study of 357 students (112 males, 245 females) responding to an online survey at a Midwestern liberal arts college revealed that males and children from divorced families perceived themselves as more promiscuous and drank more than did students from intact families. However, a significant interaction between the gender of the students and the…

  15. Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of chemodiversity in Nature (2010 JGI User Meeting)

    SciTech Connect

    Noel, Joseph

    2010-03-26

    Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting

  16. The Search for Elusive Structure: A Promiscuous Realist Case for Researching Specific Psychotic Experiences Such as Hallucinations

    PubMed Central

    Bentall, Richard P.

    2014-01-01

    Problems in psychiatric classification have impeded research into psychopathology for more than a century. Here, I briefly review several new approaches to solving this problem, including the internalizing-externalizing-psychosis spectra, the 5-factor model of psychotic symptoms, and the more recent network approach. Researchers and clinicians should probably adopt an attitude of promiscuous realism and assume that a single classification system is unlikely to be effective for all purposes, and that different systems will need to be chosen for research into etiology, public mental health research, and clinical activities. Progress in understanding the risk factors and mechanisms that lead to psychopathology is most likely to be achieved by focusing on specific types of experience or symptoms such as hallucinations. PMID:24936080

  17. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs. PMID:26748983

  18. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

    SciTech Connect

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl; Woolard, Frank; Ockey, Denise; McPhee, Derek; Renninger, Neil; Chang, Michelle; Baker, David; Keasling, Jay

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

  19. 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.

    PubMed

    Pinto, Sheena; Stark, Hans-Jürgen; Martin, Iris; Boukamp, Petra; Kyewski, Bruno

    2015-01-01

    Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs. PMID:26275017

  20. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

    PubMed Central

    Dutta, Mouparna; Ghosh, Anindya S.; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J.; Dandekar, Abhaya M.; Goñi, Félix M.

    2015-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  1. Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

    PubMed Central

    Zhang, Yu; An, Jiao; Ye, Wei; Yang, Guangyu; Qian, Zhi-Gang; Chen, Hai-Feng; Cui, Li

    2012-01-01

    The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds. PMID:22798358

  2. A Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically Promiscuous Enzyme

    PubMed Central

    McWhirter, Claire; Lund, Elizabeth A.; Tanifum, Eric A.; Feng, Guoqiang; Sheikh, Qaiser; Hengge, Alvan C.; Williams, Nicholas H.

    2009-01-01

    The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKas of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted βlg of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are 18(V/K)bridge = 1.0170 and 15(V/K) = 1.0010 which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted βlg of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the βlg measured for monoester substrates, indicative of similar transition states. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction, and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates. PMID:18798625

  3. Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

    PubMed Central

    Obembe, Olawole O.; Jacobsen, Evert; Timmers, Jaap; Gilbert, Harry; Blake, Anthony W.; Knox, J. Paul; Visser, Richard G. F.

    2007-01-01

    We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth. PMID:17622484

  4. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

    PubMed

    Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

    2016-06-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. PMID:27048590

  5. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    PubMed Central

    Costa, Sara R.; Marek, Magdalena; Axelsen, Kristian B.; Theorin, Lisa; Pomorski, Thomas G.; López-Marqués, Rosa L.

    2016-01-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. PMID:27048590

  6. An unexpected promiscuous activity of 4-oxalocrotonate tautomerase: the cis-trans isomerisation of nitrostyrene.

    PubMed

    Zandvoort, Ellen; Geertsema, Edzard M; Baas, Bert-Jan; Quax, Wim J; Poelarends, Gerrit J

    2012-09-01

    Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k(cat) /K(m) = 1.9×10(3)  M(-1)  s(-1) ). PMID:22851288

  7. Promiscuous Speciation with Gene Flow in Silverside Fish Genus Odontesthes (Atheriniformes, Atherinopsidae) from South Western Atlantic Ocean Basins

    PubMed Central

    García, Graciela; Ríos, Néstor; Gutiérrez, Verónica; Varela, Jorge Guerra; Bouza Fernández, Carmen; Pardo, Belén Gómez; Portela, Paulino Martínez

    2014-01-01

    The present paper integrates phylogenetic and population genetics analyses based on mitochondrial and nuclear molecular markers in silversides, genus Odontesthes, from a non-sampled area in the SW Atlantic Ocean to address species discrimination and to define Managements Units for sustainable conservation. All phylogenetic analyses based on the COI mitochondrial gene were consistent to support the monophyly of the genus Odontesthes and to include O. argentinensis, O. perugiae-humensis and some O. bonariensis haplotypes in a basal polytomy conforming a major derivative clade. Microsatellites data revealed somewhat higher genetic variability values in the O. argentinensis-perugia populations than in O. bonariensis and O. perugia-humensis taxa. Contrasting population genetics structuring emerged from mitochondrial and microsatellites analyses in these taxa. Whereas mitochondrial data supported two major groups (O. argentinensis-perugia-humensis vs. O. bonariensis-perugiae-humensis populations), microsatellite data detected three major genetic entities represented by O. bonariensis, O. perugiae-humensis and an admixture of populations belonging to O. argentinensis-perugiae respectively. Therefore, the star COI polytomy in the tree topology involving these taxa could be interpreted by several hypothetic scenarios such as the existence of shared ancestral polymorphisms, incomplete lineage sorting in a radiating speciation process and/or reticulation events. Present findings support that promiscuous and recent contact between incipient species sharing asymmetric gene flow exchanges, blurs taxa boundaries yielding complicated taxonomy and Management Units delimitation in silverside genus Odontesthes from SW Atlantic Ocean basins. PMID:25126842

  8. Persistent infection and promiscuous recombination of multiple genotypes of an RNA virus within a single host generate extensive diversity.

    PubMed

    Weng, Ziming; Barthelson, Roger; Gowda, Siddarame; Hilf, Mark E; Dawson, William O; Galbraith, David W; Xiong, Zhongguo

    2007-01-01

    Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses. PMID:17878952

  9. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

    PubMed

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  10. Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

    PubMed Central

    Boer, D. Roeland; Ruiz-Masó, José Angel; Rueda, Manuel; Petoukhov, Maxim V.; Machón, Cristina; Svergun, Dmitri I.; Orozco, Modesto; del Solar, Gloria; Coll, Miquel

    2016-01-01

    DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation. PMID:26875695

  11. Structure and regulatory targets of SCO3201, a highly promiscuous TetR-like regulator of Streptomyces coelicolor M145.

    PubMed

    Xu, Delin; Waack, Paul; Zhang, Qizhong; Werten, Sebastiaan; Hinrichs, Winfried; Virolle, Marie-Joelle

    2014-07-18

    SCO3201, a regulator of the TetR family, is a strong repressor of both morphological differentiation and antibiotic production when overexpressed in Streptomyces coelicolor. Here, we report the identification of 14 novel putative regulatory targets of this regulator using in vitro formaldehyde cross-linking. Direct binding of purified His6-SCO3201 was demonstrated for the promoter regions of 5 regulators (SCO1716, SCO1950, SCO3367, SCO4009 and SCO5046), a putative histidine phosphatase (SCO1809), an acetyltransferase (SCO0988) and the polyketide synthase RedX (SCO5878), using EMSA. Reverse transcriptional analysis demonstrated that the expression of the transcriptional regulators SCO1950, SCO4009, SCO5046, as well as of SCO0988 and RedX was down regulated, upon SCO3201 overexpression, whereas the expression of SCO1809 and SCO3367 was up regulated. A consensus binding motif was derived via alignment of the promoter regions of the genes negatively regulated. The positions of the predicted operator sites were consistent with a direct repressive effect of SCO3201 on 5 out of 7 of these promoters. Furthermore, the 2.1Å crystal structure of SCO3201 was solved, which provides a possible explanation for the high promiscuity of this regulator that might account for its dramatic effect on the differentiation process of S. coelicolor. PMID:24928397

  12. A dynamical model of the adaptive immune system: effects of cells promiscuity, antigens and B-B interactions

    NASA Astrophysics Data System (ADS)

    Bartolucci, Silvia; Annibale, Alessia

    2015-08-01

    We analyse a minimal model for the primary response in the adaptive immune system comprising three different players: antigens, T and B cells. We assume B-T interactions to be diluted and sampled locally from heterogeneous degree distributions, which mimic B cells receptors' promiscuity. We derive dynamical equations for the order parameters quantifying the B cells activation and study the nature and stability of the stationary solutions using linear stability analysis and Monte Carlo simulations.The system's behaviour is studied in different scaling regimes of the number of B cells, dilution in the interactions and number of antigens. Our analysis shows that: (i) B cells activation depends on the number of receptors in such a way that cells with an insufficient number of triggered receptors cannot be activated; (ii) idiotypic (i.e. B-B) interactions enhance parallel activation of multiple clones, improving the system's ability to fight different pathogens in parallel; (iii) the higher the fraction of antigens within the host the harder is for the system to sustain parallel signalling to B cells, crucial for the homeostatic control of cell numbers.

  13. The Promiscuity of [beta]-Strand Pairing Allows for Rational Design of [beta]-Sheet Face Inversion

    SciTech Connect

    Makabe, Koki; Koide, Shohei

    2009-06-17

    Recent studies suggest the dominant role of main-chain H-bond formation in specifying {beta}-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing {beta}-sheet-based nanomaterials. Here we show rational design of {beta}-sheet face inversions by incremental deletions of {beta}-strands from the single-layer {beta}-sheet of Borrelia outer surface protein A. We show that a {beta}-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a {beta}-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success of the design and supported the importance of main-chain H-bonds in determining {beta}-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on {beta}-rich peptide self-assemblies.

  14. Self-recognition drives the preferential accumulation of promiscuous CD4+ T-cells in aged mice

    PubMed Central

    Deshpande, Neha R; Parrish, Heather L; Kuhns, Michael S

    2015-01-01

    T-cell recognition of self and foreign peptide antigens presented in major histocompatibility complex molecules (pMHC) is essential for life-long immunity. How the ability of the CD4+ T-cell compartment to bind self- and foreign-pMHC changes over the lifespan remains a fundamental aspect of T-cell biology that is largely unexplored. We report that, while old mice (18–22 months) contain fewer CD4+ T-cells compared with adults (8–12 weeks), those that remain have a higher intrinsic affinity for self-pMHC, as measured by CD5 expression. Old mice also have more cells that bind individual or multiple distinct foreign-pMHCs, and the fold increase in pMHC-binding populations is directly related to their CD5 levels. These data demonstrate that the CD4+ T-cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T-cell receptor interactions with self-pMHC. DOI: http://dx.doi.org/10.7554/eLife.05949.001 PMID:26173205

  15. Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

    PubMed

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids). PMID:21673887

  16. Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter.

    PubMed

    Fraizer, G C; Wu, Y J; Hewitt, S M; Maity, T; Ton, C C; Huff, V; Saunders, G F

    1994-03-25

    The Wilms' tumor gene, WT1, is expressed in few tissues, mainly the developing kidney, genitourinary system, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kidney and hematopoietic cell lines. We found three transcription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase II promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC-rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This finding suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identified a transcriptional enhancer located 3' of the WT1 gene > 50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested. PMID:8132626

  17. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  18. MINEs: Open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics

    SciTech Connect

    Jeffryes, James G.; Colastani, Ricardo L.; Elbadawi-Sidhu, Mona; Kind, Tobias; Niehaus, Thomas D.; Broadbelt, Linda J.; Hanson, Andrew D.; Fiehn, Oliver; Tyo, Keith E. J.; Henry, Christopher S.

    2015-08-28

    Metabolomics have proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography–mass spectrometry (LC–MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases. Here we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likely to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC–MS accurate mass data enabled the identity of an unknown peak to be confidently predicted. MINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose results

  19. MINEs: Open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics

    DOE PAGESBeta

    Jeffryes, James G.; Colastani, Ricardo L.; Elbadawi-Sidhu, Mona; Kind, Tobias; Niehaus, Thomas D.; Broadbelt, Linda J.; Hanson, Andrew D.; Fiehn, Oliver; Tyo, Keith E. J.; Henry, Christopher S.

    2015-08-28

    Metabolomics have proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography–mass spectrometry (LC–MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases. Here we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likelymore » to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC–MS accurate mass data enabled the identity of an unknown peak to be confidently predicted. MINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose

  20. Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

    SciTech Connect

    Moretti, Rocco; Chang, Aram; Peltier-Pain, Pauline; Bingman, Craig A.; Phillips, Jr., George N.; Thorson, Jon S.

    2012-03-15

    Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native d- and l-sugars (both a- and b-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.

  1. Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-ACnp1 in Fission Yeast

    PubMed Central

    Choi, Eun Shik; Strålfors, Annelie; Catania, Sandra; Castillo, Araceli G.; Svensson, J. Peter; Pidoux, Alison L.; Ekwall, Karl; Allshire, Robin C.

    2012-01-01

    Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-ACnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-ACnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-ACnp1 assembly at endogenous centromeres where CENP-ACnp1 is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-ACnp1 at specific loci, including subtelomeric regions, where CENP-ACnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-ACnp1 chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-ACnp1 to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-ACnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. PMID:23028377

  2. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

    PubMed Central

    Hallberg, Zachary F.; Wang, Xin C.; Wright, Todd A.; Nan, Beiyan; Ad, Omer; Yeo, Jongchan; Hammond, Ming C.

    2016-01-01

    Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling. PMID:26839412

  3. The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

    PubMed

    Shin, Sun-Mi; Choi, Jin Myung; di Luccio, Eric; Lee, Yong-Jik; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2015-11-01

    UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism. PMID:26344854

  4. MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity.

    PubMed

    Dobó, József; Harmat, Veronika; Beinrohr, László; Sebestyén, Edina; Závodszky, Péter; Gál, Péter

    2009-07-15

    Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2, only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several non-complement substrates, like fibrinogen and factor XIII, have also been reported. MASP-1 belongs to the C1r/C1s/MASP family of modular serine proteases; however, its serine protease domain is evolutionary different. We have determined the crystal structure of the catalytic region of active MASP-1 and refined it to 2.55 A resolution. Unusual features of the structure are an internal salt bridge (similar to one in factor D) between the S1 Asp189 and Arg224, and a very long 60-loop. The functional and evolutionary differences between MASP-1 and the other members of the C1r/C1s/MASP family are reflected in the crystal structure. Structural comparison of the protease domains revealed that the substrate binding groove of MASP-1 is wide and resembles that of trypsin rather than early complement proteases explaining its relaxed specificity. Also, MASP-1's multifunctional behavior as both a complement and a coagulation enzyme is in accordance with our observation that antithrombin in the presence of heparin is a more potent inhibitor of MASP-1 than C1 inhibitor. Overall, MASP-1 behaves as a promiscuous protease. The structure shows that its substrate binding groove is accessible; however, its reactivity could be modulated by an unusually large 60-loop and an internal salt bridge involving the S1 Asp. PMID:19564340

  5. RNF17 blocks promiscuous activity of PIWI proteins in mouse testes

    PubMed Central

    Wasik, Kaja A.; Tam, Oliver H.; Knott, Simon R.; Falciatori, Ilaria; Hammell, Molly; Vagin, Vasily V.; Hannon, Gregory J.

    2015-01-01

    PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction. PMID:26115953

  6. The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for Archaeal isopentenyl phosphate kinase

    PubMed Central

    Mabanglo, Mark F.; Serohijos, Adrian W. R.; Poulter, C. Dale

    2011-01-01

    Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate (IPP) in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK, and H58 and K18 in S. wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase superfamily. We measured the fosfomycin kinase activity of THA IPK, Km = 15.1 ± 1.0 mM and kcat = (4.0 ± 0.1) × 10−2 s−1, resulting in a catalytic efficiency, kcat/Km = 2.6 M−1s−1, that is five orders of magnitude less than the native reaction. Fosfomycin is a competitive inhibitor of IPK, Ki = 3.6 ± 0.2 mM. Molecular dynamics simulation of the IPK•fosfomycin•MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK•IP•ATP complex that it engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin producing organisms. PMID:22148590

  7. Improved high-performance liquid chromatographic method for N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B) activity determination using uridine diphospho-N-acetylgalactosamine-4-sulfate.

    PubMed

    Leznicki, A J; Bialkowski, K

    1997-08-29

    UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 mumol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation; (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination < or = 3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K(m) = 85 microM. PMID:9323540

  8. An Ecological Analysis of the Effects of Deviant Peer Clustering on Sexual Promiscuity, Problem Behavior, and Childbearing from Early Adolescence to Adulthood: An Enhancement of the Life History Framework

    ERIC Educational Resources Information Center

    Dishion, Thomas J.; Ha, Thao; Veronneau, Marie-Helene

    2012-01-01

    The authors propose that peer relationships should be included in a life history perspective on adolescent problem behavior. Longitudinal analyses were used to examine deviant peer clustering as the mediating link between attenuated family ties, peer marginalization, and social disadvantage in early adolescence and sexual promiscuity in middle…

  9. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine

    PubMed Central

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A.; Dmitriev, Igor P.; Curiel, David T.; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  10. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. PMID:25552543

  11. Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.

    PubMed

    Li, Yanhong; Yu, Hai; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Qu, Jingyao; Hie, Liana; Chen, Xi

    2014-02-01

    The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts. PMID:23661084

  12. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    PubMed

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  13. The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response▿

    PubMed Central

    Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S.

    2011-01-01

    BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols. PMID:21367979

  14. Molecular diagnosis of 65 families with mucopolysaccharidosis type II (Hunter syndrome) characterized by 16 novel mutations in the IDS gene: Genetic, pathological, and structural studies on iduronate-2-sulfatase.

    PubMed

    Kosuga, Motomichi; Mashima, Ryuichi; Hirakiyama, Asami; Fuji, Naoko; Kumagai, Tadayuki; Seo, Joo-Hyun; Nikaido, Mari; Saito, Seiji; Ohno, Kazuki; Sakuraba, Hitoshi; Okuyama, Torayuki

    2016-07-01

    Mucopolysaccharidosis type II (MPS II: also called as Hunter syndrome) is an X-linked recessive lysosomal storage disorder characterized by the accumulation of extracellular glycosaminoglycans due to the deficiency of the enzyme iduronate-2-sulfatase (IDS). Previous observations suggested that MPS II can be classified into two distinct disease subtypes: (1) severe type of MPS II involves a decline in the cognitive ability of a patient and (2) attenuated type of MPS II exhibits no such intellectual phenotype. To determine whether such disease subtypes of MPS II could be explained by genetic diagnosis, we analyzed mutations in the IDS gene of 65 patients suffering from MPS II among the Japanese population who were diagnosed with both the accumulation of urinary glycosaminoglycans and a decrease in their IDS enzyme activity between 2004 and 2014. Among the specimens examined, we identified the following mutations: 33 missense, 8 nonsense, 7 frameshift, 4 intronic changes affecting splicing, 8 recombinations involving IDS-IDS2, and 7 other mutations including 4 large deletions. Consistent with the previous data, the results of our study showed that most of the attenuated phenotype was derived from the missense mutations of the IDS gene, whereas mutations associated with a large structural alteration including recombination, splicing, frameshift, and nonsense mutations were linked to the severe phenotype of MPS II. Furthermore, we conducted a homology modeling study of IDS P120R and N534I mutant as representatives of the causative mutation of the severe and attenuated type of MPS II, respectively. We found that the substitution of P120R of the IDS enzyme was predicted to deform the α-helix generated by I119-F123, leading to the major structural alteration of the wild-type IDS enzyme. In sharp contrast, the effect of the structural alteration of N534I was marginal; thus, this mutation was pathogenically predicted to be associated with the attenuated type of MPS II

  15. Mechanistic study of the radical SAM-dependent amine dehydrogenation reactions.

    PubMed

    Ji, Xinjian; Liu, Wan-Qiu; Yuan, Shuguang; Yin, Yue; Ding, Wei; Zhang, Qi

    2016-08-18

    The radical SAM enzyme NosL catalyzes the conversion of l-Trp to 3-methyl-2-indolic acid, and this reaction is initiated by the 5'-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction from the l-Trp amino group. We demonstrate here that when d-Trp was used in the NosL reaction, hydrogen abstraction occurs promiscuously at both the amino group and Cα of d-Trp. These results inspired us to establish the detailed mechanism of l-Trp amine dehydrogenation catalyzed by a NosL mutant, and to engineer a novel radical SAM-dependent l-Tyr amine dehydrogenase from the thiamine biosynthesis enzyme ThiH. PMID:27492649

  16. Drug Reactions

    MedlinePlus

    Most of the time, medicines make our lives better. They reduce aches and pains, fight infections, and control problems such as high blood pressure or diabetes. But medicines can also cause unwanted reactions. One problem is ...

  17. Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical reaction.

    PubMed

    Schenk, Gerhard; Mitić, Nataša; Gahan, Lawrence R; Ollis, David L; McGeary, Ross P; Guddat, Luke W

    2012-09-18

    Binuclear metallohydrolases are a large family of enzymes that require two closely spaced transition metal ions to carry out a plethora of hydrolytic reactions. Representatives include purple acid phosphatases (PAPs), enzymes that play a role in bone metabolism and are the only member of this family with a heterovalent binuclear center in the active form (Fe(3+)-M(2+), M = Fe, Zn, Mn). Other members of this family are urease, which contains a di-Ni(2+) center and catalyzes the breakdown of urea, arginase, which contains a di-Mn(2+) center and catalyzes the final step in the urea cycle, and the metallo-β-lactamases, which contain a di-Zn(2+) center and are virulence factors contributing to the spread of antibiotic-resistant pathogens. Binuclear metallohydrolases catalyze numerous vital reactions and are potential targets of drugs against a wide variety of human disorders including osteoporosis, various cancers, antibiotic resistance, and erectile dysfunctions. These enzymes also tend to catalyze more than one reaction. An example is an organophosphate (OP)-degrading enzyme from Enterobacter aerogenes (GpdQ). Although GpdQ is part of a pathway that is used by bacteria to degrade glycerolphosphoesters, it hydrolyzes a variety of other phosphodiesters and displays low levels of activity against phosphomono- and triesters. Such a promiscuous nature may have assisted the apparent recent evolution of some binuclear metallohydrolases to deal with situations created by human intervention such as OP pesticides in the environment. OP pesticides were first used approximately 70 years ago, and therefore the enzymes that bacteria use to degrade them must have evolved very quickly on the evolutionary time scale. The promiscuous nature of enzymes such as GpdQ makes them ideal candidates for the application of directed evolution to produce new enzymes that can be used in bioremediation and against chemical warfare. In this Account, we review the mechanisms employed by binuclear

  18. Immunogenicity of a Promiscuous T Cell Epitope Peptide Based Conjugate Vaccine against Benzo[a]pyrene: Redirecting Antibodies to the Hapten

    PubMed Central

    Schellenberger, Mario T.; Grova, Nathalie; Farinelle, Sophie; Willième, Stéphanie; Revets, Dominique; Muller, Claude P.

    2012-01-01

    The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142–51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15–56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted. PMID:22666501

  19. Immunogenicity of a promiscuous T cell epitope peptide based conjugate vaccine against benzo[a]pyrene: redirecting antibodies to the hapten.

    PubMed

    Schellenberger, Mario T; Grova, Nathalie; Farinelle, Sophie; Willième, Stéphanie; Revets, Dominique; Muller, Claude P

    2012-01-01

    The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142-51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15-56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted. PMID:22666501

  20. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

    PubMed Central

    Renata, Hans; Wang, Z. Jane

    2015-01-01

    High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

  1. In with the Old, in with the New: The Promiscuity of the Duplication Process Engenders Diverse Pathways for Novel Gene Creation

    PubMed Central

    Katju, Vaishali

    2012-01-01

    The gene duplication process has exhibited far greater promiscuity in the creation of paralogs with novel exon-intron structures than anticipated even by Ohno. In this paper I explore the history of the field, from the neo-Darwinian synthesis through Ohno's formulation of the canonical model for the evolution of gene duplicates and culminating in the present genomic era. I delineate the major tenets of Ohno's model and discuss its failure to encapsulate the full complexity of the duplication process as revealed in the era of genomics. I discuss the diverse classes of paralogs originating from both DNA- and RNA-mediated duplication events and their evolutionary potential for assuming radically altered functions, as well as the degree to which they can function unconstrained from the pressure of gene conversion. Lastly, I explore theoretical population-genetic considerations of how the effective population size (Ne) of a species may influence the probability of emergence of genes with radically altered functions. PMID:23008799

  2. A novel therapeutic strategy of lipidated promiscuous peptide against Mycobacterium tuberculosis by eliciting Th1 and Th17 immunity of host

    PubMed Central

    Rai, Pradeep K; Chodisetti, Sathi Babu; Nadeem, Sajid; Maurya, Sudeep K; Gowthaman, Uthaman; Zeng, Weiguang; Janmeja, Ashok K; Jackson, David C; Agrewala, Javed N

    2016-01-01

    Regardless of the fact that potent drug-regimen is currently available, tuberculosis continues to kill 1.5 million people annually. Tuberculosis patients are not only inflicted by the trauma of disease but they also suffer from the harmful side-effects, immune suppression and drug resistance instigated by prolonged therapy. It is an exigency to introduce radical changes in the existing drug-regime and discover safer and better therapeutic measures. Hence, we designed a novel therapeutic strategy by reinforcing the efficacy of drugs to kill Mtb by concurrently boosting host immunity by L91. L91 is chimera of promiscuous epitope of Acr1 antigen of Mtb and TLR-2 agonist Pam2Cys. The adjunct therapy using drugs and L91 (D-L91) significantly declined the bacterial load in Mtb infected animals. The mechanism involved was through enhancement of IFN-γ+TNF-α+ polyfunctional Th1 cells and IL-17A+IFN-γ+ Th17 cells, enduring memory CD4 T cells and downregulation of PD-1. The down-regulation of PD-1 prevents CD4 T cells from undergoing exhaustion and improves their function against Mtb. Importantly, the immune response observed in animals could be replicated using T cells of tuberculosis patients on drug therapy. In future, D-L91 therapy can invigorate drugs potency to treat tuberculosis patients and reduce the dose and duration of drug-regime. PMID:27052185

  3. A comparison of scent marking between a monogamous and promiscuous species of peromyscus: pair bonded males do not advertise to novel females.

    PubMed

    Becker, Elizabeth A; Petruno, Sarah; Marler, Catherine A

    2012-01-01

    Scent marking can provide behavioral and physiological information including territory ownership and mate advertisement. It is unknown how mating status and pair cohabitation influence marking by males from different social systems. We compared the highly territorial and monogamous California mouse (Peromyscus californicus) to the less territorial and promiscuous white-footed mouse (P. leucopus). Single and mated males of both species were assigned to one of the following arenas lined with filter paper: control (unscented arena), male scented (previously scent-marked by a male conspecific), or females present (containing females in small cages). As expected, the territorial P. californicus scent marked and overmarked an unfamiliar male conspecific's scent marks more frequently than P. leucopus. Species differences in responses to novel females were also found based on mating status. The presence of unfamiliar females failed to induce changes in scent marking in pair bonded P. californicus even though virgin males increased marking behavior. Pair bonding appears to reduce male advertisement for novel females. This is in contrast to P. leucopus males that continue to advertise regardless of mating status. Our data suggest that communication through scent-marking can diverge significantly between species based on mating system and that there are physiological mechanisms that can inhibit responsiveness of males to female cues. PMID:22393377

  4. Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

    PubMed Central

    Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

    2014-01-01

    Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

  5. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    PubMed

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine. PMID:23242635

  6. Unique mononuclear Mn(II) complexes of an end-off compartmental Schiff base ligand: experimental and theoretical studies on their bio-relevant catalytic promiscuity.

    PubMed

    Adhikary, Jaydeep; Chakraborty, Aratrika; Dasgupta, Sanchari; Chattopadhyay, Shyamal Kumar; Kruszynski, Rafał; Trzesowska-Kruszynska, Agata; Stepanović, Stepan; Gruden-Pavlović, Maja; Swart, Marcel; Das, Debasis

    2016-08-01

    Three new mononuclear manganese(ii) complexes, namely [Mn(HL)2]·2ClO4 (1), [Mn(HL)(N(CN)2)(H2O)2]·ClO4 (2) and [Mn(HL)(SCN)2] (3) [LH = 4-tert-butyl-2,6-bis-[(2-pyridin-2-yl-ethylimino)-methyl]-phenol], have been synthesized and structurally characterized. An "end-off" compartmental ligand (LH) possesses two symmetrical compartments with N2O binding sites but accommodates only one manganese atom instead of two due to the protonation of the imine nitrogen of one compartment. Although all three complexes are mononuclear, complex 1 is unique as it has a 1 : 2 metal to ligand stoichiometry. The catalytic promiscuity of complexes 1-3 in terms of two different bio-relevant catalytic activities namely catecholase and phenoxazinone synthase has been thoroughly investigated. EPR and cyclic voltametric studies reveal that radical formation rather than metal centered redox participation is responsible for their catecholase-like and phenoxazinone synthase-like catalytic activity. A computational approach suggests that imine bond bound radical generation rather than phenoxo radical formation is most likely responsible for the oxidizing properties of the complexes. PMID:27430642

  7. U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs.

    PubMed

    Jenkins, Jermaine L; Agrawal, Anant A; Gupta, Ankit; Green, Michael R; Kielkopf, Clara L

    2013-04-01

    Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65). PMID:23376934

  8. Drug Reactions

    MedlinePlus

    ... using any of these products. Some types of food may also cause adverse drug reactions. For example, grapefruit and grapefruit juice, as well as alcohol and caffeine, may affect how drugs work. Every time your doctor ... interactions with any foods or beverages. What about medicines I've used ...

  9. Characterization of Promiscuous Binding of Phosphor Ligands to Breast-Cancer-Gene 1 (BRCA1) C-Terminal (BRCT): Molecular Dynamics, Free Energy, Entropy and Inhibitor Design.

    PubMed

    You, Wanli; Huang, Yu-Ming M; Kizhake, Smitha; Natarajan, Amarnath; Chang, Chia-En A

    2016-08-01

    Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain. Several studies identified short phosphopeptides as tight BRCT binders. Here we investigated the thermodynamic properties of 18 phosphopeptides or peptide with phosphate mimic and three compounds with phosphate groups binding to BRCT to understand promiscuous molecular recognition and guide inhibitor design. We performed molecular dynamics (MD) simulations to investigate the interactions between inhibitors and BRCT and their dynamic behavior in the free and bound states. MD simulations revealed the key role of loops in altering the shape and size of the binding site to fit various ligands. The mining minima (M2) method was used for calculating binding free energy to explore the driving forces and the fine balance between configuration entropy loss and enthalpy gain. We designed a rigidified ligand, which showed unfavorable experimental binding affinity due to weakened enthalpy. This was because it lacked the ability to rearrange itself upon binding. Investigation of another phosphate group containing compound, C1, suggested that the entropy loss can be reduced by preventing significant narrowing of the energy well and introducing multiple new compound conformations in the bound states. From our computations, we designed an analog of C1 that introduced new intermolecular interactions to strengthen attractions while maintaining small entropic penalty. This study shows that flexible compounds do not always encounter larger entropy penalty, compared with other more rigid binders, and highlights a new strategy for inhibitor design. PMID

  10. Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2001-12-11

    The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity. PMID:11732894

  11. Factors that promote H3 chromatin integrity during transcription prevent promiscuous deposition of CENP-A(Cnp1) in fission yeast.

    PubMed

    Choi, Eun Shik; Strålfors, Annelie; Catania, Sandra; Castillo, Araceli G; Svensson, J Peter; Pidoux, Alison L; Ekwall, Karl; Allshire, Robin C

    2012-09-01

    Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. PMID:23028377

  12. Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases.

    PubMed

    Li, Yanhong; Xue, Mengyang; Sheng, Xue; Yu, Hai; Zeng, Jie; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Wang, Peng G; Chen, Xi

    2016-04-15

    β1-3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1-4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1-3Galβ1-4GlcβProN3 and Galβ1-3Galβ1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents. PMID:26968649

  13. Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents – real hits or promiscuous artifacts?

    PubMed Central

    Johnston, Paul A.

    2010-01-01

    Redox cycling compounds (RCCs) generate µM concentrations of hydrogen peroxide (H2O2) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H2O2 generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H2O2-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H2O2 in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H2O2 as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds which can generate intracellular H2O2 by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; critical resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs. PMID:21075044

  14. New Tricks for “Old” Domains: How Novel Architectures and Promiscuous Hubs Contributed to the Organization and Evolution of the ECM

    PubMed Central

    Cromar, Graham; Wong, Ka-Chun; Loughran, Noeleen; On, Tuan; Song, Hongyan; Xiong, Xuejian; Zhang, Zhaolei; Parkinson, John

    2014-01-01

    The extracellular matrix (ECM) is a defining characteristic of metazoans and consists of a meshwork of self-assembling, fibrous proteins, and their functionally related neighbours. Previous studies, focusing on a limited number of gene families, suggest that vertebrate complexity predominantly arose through the duplication and subsequent modification of retained, preexisting ECM genes. These genes provided the structural underpinnings to support a variety of specialized tissues, as well as a platform for the organization of spatio-temporal signaling and cell migration. However, the relative contributions of ancient versus novel domains to ECM evolution have not been quantified across the full range of ECM proteins. Here, utilizing a high quality list comprising 324 ECM genes, we reveal general and clade-specific domain combinations, identifying domains of eukaryotic and metazoan origin recruited into new roles in approximately two-third of the ECM proteins in humans representing novel vertebrate proteins. We show that, rather than acquiring new domains, sampling of new domain combinations has been key to the innovation of paralogous ECM genes during vertebrate evolution. Applying a novel framework for identifying potentially important, noncontiguous, conserved arrangements of domains, we find that the distinct biological characteristics of the ECM have arisen through unique evolutionary processes. These include the preferential recruitment of novel domains to existing architectures and the utilization of high promiscuity domains in organizing the ECM network around a connected array of structural hubs. Our focus on ECM proteins reveals that distinct types of proteins and/or the biological systems in which they operate have influenced the types of evolutionary forces that drive protein innovation. This emphasizes the need for rigorously defined systems to address questions of evolution that focus on specific systems of interacting proteins. PMID:25323955

  15. Characterization of Promiscuous Binding of Phosphor Ligands to Breast-Cancer-Gene 1 (BRCA1) C-Terminal (BRCT): Molecular Dynamics, Free Energy, Entropy and Inhibitor Design

    PubMed Central

    Huang, Yu-ming M.; Kizhake, Smitha; Natarajan, Amarnath; Chang, Chia-en A.

    2016-01-01

    Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain. Several studies identified short phosphopeptides as tight BRCT binders. Here we investigated the thermodynamic properties of 18 phosphopeptides or peptide with phosphate mimic and three compounds with phosphate groups binding to BRCT to understand promiscuous molecular recognition and guide inhibitor design. We performed molecular dynamics (MD) simulations to investigate the interactions between inhibitors and BRCT and their dynamic behavior in the free and bound states. MD simulations revealed the key role of loops in altering the shape and size of the binding site to fit various ligands. The mining minima (M2) method was used for calculating binding free energy to explore the driving forces and the fine balance between configuration entropy loss and enthalpy gain. We designed a rigidified ligand, which showed unfavorable experimental binding affinity due to weakened enthalpy. This was because it lacked the ability to rearrange itself upon binding. Investigation of another phosphate group containing compound, C1, suggested that the entropy loss can be reduced by preventing significant narrowing of the energy well and introducing multiple new compound conformations in the bound states. From our computations, we designed an analog of C1 that introduced new intermolecular interactions to strengthen attractions while maintaining small entropic penalty. This study shows that flexible compounds do not always encounter larger entropy penalty, compared with other more rigid binders, and highlights a new strategy for inhibitor design. PMID

  16. Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

    PubMed

    Al-Attiyah, R; Mustafa, A S

    2004-01-01

    The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy. PMID:14723617

  17. Minor modifications of the C-terminal helix reschedule the favored chemical reactions catalyzed by theta class glutathione transferase T1-1.

    PubMed

    Shokeer, Abeer; Mannervik, Bengt

    2010-02-19

    Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme. PMID:20022951

  18. First Principles Modeling of Bimolecular Reactions with Diffusion

    NASA Astrophysics Data System (ADS)

    Hansen, S. K.; Scher, H.; Berkowitz, B.

    2013-12-01

    We consider three approaches to modeling A + B → C irreversible reactions in natural media: 1) a discretized diffusion-reaction equation (DRE), 2) a particle tracking (PT) scheme in which reaction occurs if and only if an A and B particle pair are within a fixed distance, r (the "reaction radius"), and 3) a PT scheme using an alternative to the fixed reaction radius: a collocation probability distribution derived directly from first principles. Each approach has advantages. In some cases a discretized DRE may be the most computationally efficient method. For PT simulations, robust codes exist based on use of a fixed reaction radius. And finally, collocation probabilities may be derived directly from the Fick's Law constant, D, which is a well-established property for most species. In each approach, a single parameter governs the 'promiscuity' of the reaction (i.e. the thermodynamic favorability of reaction, predicated on the particles being locally well mixed). For the DRE, fixed-reaction-radius PT, and collocation-based PT, these parameters are, respectively: a second-order decay rate, r, and D. We established a number of new results enhancing these approaches and relating them to each other (and to nature). In particular, a thought experiment concerning a simple system in which the predictions of each approach can be computed analytically was used to derive formulas establishing a universal one-to-one correspondence among each of the governing parameters. We thus showed the conditions for equivalence of the three approaches, and grounded both the DRE approach and the fixed-radius PT approach in the Fick's Law D. We further showed that the existing collocation-based PT theory is based on a probability distribution that is only correct for infinitesimally small times, but which can be modified to be accurate for larger times by means of continuous time random walk analysis and first-passage probability distributions. Finally, we employed a novel mathematical

  19. Probabilistic inference of biochemical reactions in microbial communities from metagenomic sequences.

    PubMed

    Jiao, Dazhi; Ye, Yuzhen; Tang, Haixu

    2013-01-01

    Shotgun metagenomics has been applied to the studies of the functionality of various microbial communities. As a critical analysis step in these studies, biological pathways are reconstructed based on the genes predicted from metagenomic shotgun sequences. Pathway reconstruction provides insights into the functionality of a microbial community and can be used for comparing multiple microbial communities. The utilization of pathway reconstruction, however, can be jeopardized because of imperfect functional annotation of genes, and ambiguity in the assignment of predicted enzymes to biochemical reactions (e.g., some enzymes are involved in multiple biochemical reactions). Considering that metabolic functions in a microbial community are carried out by many enzymes in a collaborative manner, we present a probabilistic sampling approach to profiling functional content in a metagenomic dataset, by sampling functions of catalytically promiscuous enzymes within the context of the entire metabolic network defined by the annotated metagenome. We test our approach on metagenomic datasets from environmental and human-associated microbial communities. The results show that our approach provides a more accurate representation of the metabolic activities encoded in a metagenome, and thus improves the comparative analysis of multiple microbial communities. In addition, our approach reports likelihood scores of putative reactions, which can be used to identify important reactions and metabolic pathways that reflect the environmental adaptation of the microbial communities. Source code for sampling metabolic networks is available online at http://omics.informatics.indiana.edu/mg/MetaNetSam/. PMID:23555216

  20. Theoretical study of the reaction mechanism of phenolic acid decarboxylase.

    PubMed

    Sheng, Xiang; Lind, Maria E S; Himo, Fahmi

    2015-12-01

    The cofactor-free phenolic acid decarboxylases (PADs) catalyze the non-oxidative decarboxylation of phenolic acids to their corresponding p-vinyl derivatives. Phenolic acids are toxic to some organisms, and a number of them have evolved the ability to transform these compounds, including PAD-catalyzed reactions. Since the vinyl derivative products can be used as polymer precursors and are also of interest in the food-processing industry, PADs might have potential applications as biocatalysts. We have investigated the detailed reaction mechanism of PAD from Bacillus subtilis using quantum chemical methodology. A number of different mechanistic scenarios have been considered and evaluated on the basis of their energy profiles. The calculations support a mechanism in which a quinone methide intermediate is formed by protonation of the substrate double bond, followed by C-C bond cleavage. A different substrate orientation in the active site is suggested compared to the literature proposal. This suggestion is analogous to other enzymes with p-hydroxylated aromatic compounds as substrates, such as hydroxycinnamoyl-CoA hydratase-lyase and vanillyl alcohol oxidase. Furthermore, on the basis of the calculations, a different active site residue compared to previous proposals is suggested to act as the general acid in the reaction. The mechanism put forward here is consistent with the available mutagenesis experiments and the calculated energy barrier is in agreement with measured rate constants. The detailed mechanistic understanding developed here might be extended to other members of the family of PAD-type enzymes. It could also be useful to rationalize the recently developed alternative promiscuous reactivities of these enzymes. PMID:26408050

  1. AGXT2: a promiscuous aminotransferase

    PubMed Central

    Rodionov, Roman N.; Jarzebska, Natalia; Weiss, Norbert; Lentz, Steven R.

    2014-01-01

    Alanine-glyoxylate aminotransferase 2 (AGXT2) is a multifunctional mitochondrial aminotransferase that was first identified in 1978. The physiological importance of AGXT2 was largely overlooked for three decades because AGXT2 is less active in glyoxylate metabolism than AGXT1, the enzyme that is deficient in primary hyperoxaluria type I. Recently, several novel functions of AGXT2 have been “rediscovered” in the setting of modern genomic and metabolomic studies. It is now apparent that AGXT2 has multiple substrates and products and that altered AGXT2 activity may contribute to the pathogenesis of cardiovascular, renal, neurological and hematological diseases. This article reviews the biochemical properties and physiological functions of AGXT2, its unique role at the intersection of key mitochondrial pathways, and its potential as a drug target. PMID:25294000

  2. Faculty Consulting: Responsibility or Promiscuity?

    ERIC Educational Resources Information Center

    Boyer, Carol M; Lewis, Darrell R.

    1984-01-01

    The potential benefits--to the individual, the institution, and society--and the potential costs of faculty consulting are examined. A review of the relevant literature and data precedes a presentation of new findings and a taxonomy for developing institutional guidelines. (Author/MLW)

  3. The Glyoxal Clock Reaction

    ERIC Educational Resources Information Center

    Ealy, Julie B.; Negron, Alexandra Rodriguez; Stephens, Jessica; Stauffer, Rebecca; Furrow, Stanley D.

    2007-01-01

    Research on the glyoxal clock reaction has led to adaptation of the clock reaction to a general chemistry experiment. This particular reaction is just one of many that used formaldehyde in the past. The kinetics of the glyoxal clock makes the reaction suitable as a general chemistry lab using a Calculator Based Laboratory (CBL) or a LabPro. The…

  4. Practice Gaps: Drug Reactions.

    PubMed

    Wolverton, Stephen E

    2016-07-01

    The term "drug reactions" is relevant to dermatology in three categories of reactions: cutaneous drug reactions without systemic features, cutaneous drug reactions with systemic features, and systemic drugs prescribed by the dermatologist with systematic adverse effects. This article uses examples from each of these categories to illustrate several important principles central to drug reaction diagnosis and management. The information presented will help clinicians attain the highest possible level of certainty before making clinical decisions. PMID:27363888

  5. Microfluidic chemical reaction circuits

    DOEpatents

    Lee, Chung-cheng; Sui, Guodong; Elizarov, Arkadij; Kolb, Hartmuth C.; Huang, Jiang; Heath, James R.; Phelps, Michael E.; Quake, Stephen R.; Tseng, Hsian-rong; Wyatt, Paul; Daridon, Antoine

    2012-06-26

    New microfluidic devices, useful for carrying out chemical reactions, are provided. The devices are adapted for on-chip solvent exchange, chemical processes requiring multiple chemical reactions, and rapid concentration of reagents.

  6. Continuous detonation reaction engine

    NASA Technical Reports Server (NTRS)

    Lange, O. H.; Stein, R. J.; Tubbs, H. E.

    1968-01-01

    Reaction engine operates on the principles of a controlled condensed detonation rather than on the principles of gas expansion. The detonation results in reaction products that are expelled at a much higher velocity.

  7. Allergic reactions (image)

    MedlinePlus

    Allergic reaction is a sensitivity to a specific substance, called an allergen, that is contacted through the skin, inhaled into the lungs, swallowed or injected. The body's reaction to an allergen can be mild, such as ...

  8. Allergic reactions (image)

    MedlinePlus

    Allergic reaction can be provoked by skin contact with poison plants, chemicals and animal scratches, as well as by ... dust, nuts and shellfish, may also cause allergic reaction. Medications such as penicillin and other antibiotics are ...

  9. Microscale Thermite Reactions.

    ERIC Educational Resources Information Center

    Arnaiz, Francisco J.; Aguado, Rafael; Arnaiz, Susana

    1998-01-01

    Describes the adaptation of thermite (aluminum with metal oxides) reactions from whole-class demonstrations to student-run micro-reactions. Lists detailed directions and possible variations of the experiment. (WRM)

  10. Reaction coordinates for electron transfer reactions

    SciTech Connect

    Rasaiah, Jayendran C.; Zhu Jianjun

    2008-12-07

    The polarization fluctuation and energy gap formulations of the reaction coordinate for outer sphere electron transfer are linearly related to the constant energy constraint Lagrangian multiplier m in Marcus' theory of electron transfer. The quadratic dependence of the free energies of the reactant and product intermediates on m and m+1, respectively, leads to similar dependence of the free energies on the reaction coordinates and to the same dependence of the activation energy on the reorganization energy and the standard reaction free energy. Within the approximations of a continuum model of the solvent and linear response of the longitudinal polarization to the electric field in Marcus' theory, both formulations of the reaction coordinate are expected to lead to the same results.

  11. Catalytic diastereoselective petasis reactions.

    PubMed

    Muncipinto, Giovanni; Moquist, Philip N; Schreiber, Stuart L; Schaus, Scott E

    2011-08-22

    Multicomponent Petasis reactions: the first diastereoselective Petasis reaction catalyzed by chiral biphenols that enables the synthesis of syn and anti β-amino alcohols in pure form has been developed. The reaction exploits a multicomponent approach that involves boronates, α-hydroxy aldehydes, and amines. PMID:21751322

  12. Reaction efficiency effects on binary chemical reactions

    NASA Astrophysics Data System (ADS)

    Lazaridis, Filippos; Savara, Aditya; Argyrakis, Panos

    2014-09-01

    We study the effect of the variation of reaction efficiency in binary reactions. We use the well-known A + B → 0 model, which has been extensively studied in the past. We perform simulations on this model where we vary the efficiency of reaction, i.e., when two particles meet they do not instantly react, as has been assumed in previous studies, but they react with a probability γ, where γ is in the range 0 < γ < 1. Our results show that at small γ values the system is reaction limited, but as γ increases it crosses over to a diffusion limited behavior. At early times, for small γ values, the particle density falls slower than for larger γ values. This fall-off goes over a crossover point, around the value of γ = 0.50 for high initial densities. Under a variety of conditions simulated, we find that the crossover point was dependent on the initial concentration but not on the lattice size. For intermediate and long times simulations, all γ values (in the depleted reciprocal density versus time plot) converge to the same behavior. These theoretical results are useful in models of epidemic reactions and epidemic spreading, where a contagion from one neighbor to the next is not always successful but proceeds with a certain probability, an analogous effect with the reaction probability examined in the current work.

  13. Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily*

    PubMed Central

    Miles, Zachary D.; Roberts, Sue A.; McCarty, Reid M.; Bandarian, Vahe

    2014-01-01

    6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds. PMID:24990950

  14. Discovery of a Promiscuous Non-Heme Iron Halogenase in Ambiguine Alkaloid Biogenesis: Implication for an Evolvable Enzyme Family for Late-Stage Halogenation of Aliphatic Carbons in Small Molecules.

    PubMed

    Hillwig, Matthew L; Zhu, Qin; Ittiamornkul, Kuljira; Liu, Xinyu

    2016-05-01

    The elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non-heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late-stage aliphatic C-H group functionalization. Cross-comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C-terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild-type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole-type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late-stage modification of unactivated aliphatic carbon centers in small molecules with halogens. PMID:27027281

  15. μ-Theraphotoxin-An1a: primary structure determination and assessment of the pharmacological activity of a promiscuous anti-insect toxin from the venom of the tarantula Acanthoscurria natalensis (Mygalomorphae, Theraphosidae).

    PubMed

    Rates, Breno; Prates, Maura V; Verano-Braga, Thiago; da Rocha, Angela P; Roepstorff, Peter; Borges, Carlos L; Lapied, Bruno; Murillo, Laurence; Pimenta, Adriano M C; Biondi, Ilka; De Lima, Maria Elena

    2013-08-01

    Tarantulas are included in the mygalomorph spider family Theraphosidae. Although the pharmacological diversity of theraphosid toxins (theraphotoxins) is broad, studies dedicated to the characterization of biologically active molecules from the theraphosid genus Acanthoscurria have been restricted to the investigation of antimicrobial peptides and polyamines produced by the hemocytes of Acanthoscurria gomesiana. The present study reports the purification, primary structure determination and electrophysiological effects of an anti-insect toxin, named μ-theraphotoxin-An1a (μ-TRTX-An1a), from the venom of Acanthoscurria natalensis - a tarantula species occurring in the Brazilian biomes caatinga and cerrado. The analysis of the primary structure of μ-TRTX-An1a revealed the similarity of this toxin to theraphosid toxins bearing a huwentoxin-II-like fold. Electrophysiological experiments showed that μ-TRTX-An1a (100 nM) induces membrane depolarization, increases the spontaneous firing frequency and reduces spike amplitude of cockroach dorsal unpaired median (DUM) neurons. In addition, under voltage-clamp conditions, μ-TRTX-An1a (100 nM) only partially blocks voltage-dependent sodium current amplitudes in DUM neurons without any effect on their voltage dependence. This effect correlates well with the reduction of the spontaneous action potential amplitudes. Altogether, these last results suggest that μ-TRTX-An1a affects insect neuronal voltage-dependent sodium channels, which are among possible channels targeted by this promiscuous toxin. PMID:23651762

  16. Anaphylactic reactions to cinoxacin.

    PubMed Central

    Stricker, B. H.; Slagboom, G.; Demaeseneer, R.; Slootmaekers, V.; Thijs, I.; Olsson, S.

    1988-01-01

    During 1981 to mid-1988 three cases of anaphylactic shock after treatment with the quinolone derivative cinoxacin were reviewed by the Netherlands Centre for Monitoring of Adverse Reactions to Drugs and 17 cases of an anaphylactic type of reaction notified to the World Health Organisation Collaborating Centre for International Drug Monitoring. In five out of six patients for whom data were available the reaction began shortly after taking a single capsule of a second or next course of treatment. Cinoxacin is related to nalidixic acid, and one patient previously treated with that agent subsequently had an anaphylactoid reaction to cinoxacin and later developed a skin reaction to nalidixic acid. There were no deaths, and patients treated as an emergency with plasma expanders or with adrenaline and corticosteroids generally recovered promptly and uneventfully. In view of the potentially fatal consequences of anaphylactic reactions to cinoxacin and other quinolones doctors should take care when prescribing these drugs. PMID:3147004

  17. Reactions to radiocontrast media.

    PubMed

    Hong, Sandra J; Wong, Johnson T; Bloch, Kurt J

    2002-01-01

    Adverse reactions to radiocontrast media (RCM) occur unexpectedly and may be life-threatening. This article describes an anaphylactoid reaction (AR) in one patient. The term AR refers to a syndrome clinically similar to anaphylaxis, but these reactions are independent of immunoglobulin E antibody-mediated mast cell or basophil degranulation. This article briefly reviews the literature regarding RCMs and types of reactions to RCM. The risk factors for AR to RCM infusions will be discussed along with current concepts of the pathogenesis of RCM-induced ARs. This article also describes the therapeutic management of patients who have had a previous adverse reaction to RCM and provides an approach to patients who have breakthrough reactions despite adequate premedication, but require additional radiographic studies. PMID:12476546

  18. Noncanonical Reactions of Flavoenzymes

    PubMed Central

    Sobrado, Pablo

    2012-01-01

    Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates. PMID:23203060

  19. Mechanisms in Knockout Reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-06-01

    We report the first detailed study of the relative importance of the stripping and diffraction mechanisms involved in nucleon knockout reactions, by the use of a coincidence measurement of the residue and fast proton following one-proton knockout reactions. The measurements used the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results for the reactions Be9(C9,B8+X)Y and Be9(B8,Be7+X)Y are presented and compared with theoretical predictions for the two reaction mechanisms calculated using the eikonal model. The data show a clear distinction between the stripping and diffraction mechanisms and the measured relative proportions are very well reproduced by the reaction theory. This agreement adds support to the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes.

  20. Sleeve reaction chamber system

    SciTech Connect

    Northrup, M. Allen; Beeman, Barton V.; Benett, William J.; Hadley, Dean R.; Landre, Phoebe; Lehew, Stacy L.; Krulevitch, Peter A.

    2009-08-25

    A chemical reaction chamber system that combines devices such as doped polysilicon for heating, bulk silicon for convective cooling, and thermoelectric (TE) coolers to augment the heating and cooling rates of the reaction chamber or chambers. In addition the system includes non-silicon-based reaction chambers such as any high thermal conductivity material used in combination with a thermoelectric cooling mechanism (i.e., Peltier device). The heat contained in the thermally conductive part of the system can be used/reused to heat the device, thereby conserving energy and expediting the heating/cooling rates. The system combines a micromachined silicon reaction chamber, for example, with an additional module/device for augmented heating/cooling using the Peltier effect. This additional module is particularly useful in extreme environments (very hot or extremely cold) where augmented heating/cooling would be useful to speed up the thermal cycling rates. The chemical reaction chamber system has various applications for synthesis or processing of organic, inorganic, or biochemical reactions, including the polymerase chain reaction (PCR) and/or other DNA reactions, such as the ligase chain reaction.

  1. Metal-mullite reactions

    SciTech Connect

    Loehman, R.E.; Tomsia, A.P.

    1993-11-01

    Mullite was reacted with pure Al and with Ti or Zr dissolved in Ag-Cu eutectic alloys at 1100 C in Ar. Analysis of the Ti and Zr-containing specimens showed reaction zones with compositions of Ti{sub 50}Cu{sub 3O}O{sub 20} and ZrO{sub 2}, respectively. The Al-mullite specimen showed much more extensive penetration into the ceramic and a more diffuse reaction zone than the other two systems. Al{sub 2}O{sub 3} and Si were the main reaction products for Al-mullite reaction.

  2. Systematic Analysis of the Associations between Adverse Drug Reactions and Pathways

    PubMed Central

    Chen, Xiaowen; Wang, Yanqiu; Wang, Pingping; Lian, Baofeng; Li, Chunquan; Wang, Jing; Li, Xia; Jiang, Wei

    2015-01-01

    Adverse drug reactions (ADRs) are responsible for drug candidate failure during clinical trials. It is crucial to investigate biological pathways contributing to ADRs. Here, we applied a large-scale analysis to identify overrepresented ADR-pathway combinations through merging clinical phenotypic data, biological pathway data, and drug-target relations. Evaluation was performed by scientific literature review and defining a pathway-based ADR-ADR similarity measure. The results showed that our method is efficient for finding the associations between ADRs and pathways. To more systematically understand the mechanisms of ADRs, we constructed an ADR-pathway network and an ADR-ADR network. Through network analysis on biology and pharmacology, it was found that frequent ADRs were associated with more pathways than infrequent and rare ADRs. Moreover, environmental information processing pathways contributed most to the observed ADRs. Integrating the system organ class of ADRs, we found that most classes tended to interact with other classes instead of themselves. ADR classes were distributed promiscuously in all the ADR cliques. These results reflected that drug perturbation to a certain pathway can cause changes in multiple organs, rather than in one specific organ. Our work not only provides a global view of the associations between ADRs and pathways, but also is helpful to understand the mechanisms of ADRs. PMID:26495310

  3. Hydrogen evolution reaction catalyst

    DOEpatents

    Subbaraman, Ram; Stamenkovic, Vojislav; Markovic, Nenad; Tripkovic, Dusan

    2016-02-09

    Systems and methods for a hydrogen evolution reaction catalyst are provided. Electrode material includes a plurality of clusters. The electrode exhibits bifunctionality with respect to the hydrogen evolution reaction. The electrode with clusters exhibits improved performance with respect to the intrinsic material of the electrode absent the clusters.

  4. Chemical Reaction Problem Solving.

    ERIC Educational Resources Information Center

    Veal, William

    1999-01-01

    Discusses the role of chemical-equation problem solving in helping students predict reaction products. Methods for helping students learn this process must be taught to students and future teachers by using pedagogical skills within the content of chemistry. Emphasizes that solving chemical reactions should involve creative cognition where…

  5. Applications of Reaction Rate

    ERIC Educational Resources Information Center

    Cunningham, Kevin

    2007-01-01

    This article presents an assignment in which students are to research and report on a chemical reaction whose increased or decreased rate is of practical importance. Specifically, students are asked to represent the reaction they have chosen with an acceptable chemical equation, identify a factor that influences its rate and explain how and why it…

  6. REUSABLE REACTION VESSEL

    DOEpatents

    Soine, T.S.

    1963-02-26

    This patent shows a reusable reaction vessel for such high temperature reactions as the reduction of actinide metal chlorides by calcium metal. The vessel consists of an outer metal shell, an inner container of refractory material such as sintered magnesia, and between these, a bed of loose refractory material impregnated with thermally conductive inorganic salts. (AEC)

  7. Nuclear Reaction Data Centers

    SciTech Connect

    McLane, V.; Nordborg, C.; Lemmel, H.D.; Manokhin, V.N.

    1988-01-01

    The cooperating Nuclear Reaction Data Centers are involved in the compilation and exchange of nuclear reaction data for incident neutrons, charged particles and photons. Individual centers may also have services in other areas, e.g., evaluated data, nuclear structure and decay data, reactor physics, nuclear safety; some of this information may also be exchanged between interested centers. 20 refs., 1 tab.

  8. Degradations and Rearrangement Reactions

    NASA Astrophysics Data System (ADS)

    Zhang, Jianbo

    This section deals with recent reports concerning degradation and rearrangement reactions of free sugars as well as some glycosides. The transformations are classified in chemical and enzymatic ways. In addition, the Maillard reaction will be discussed as an example of degradation and rearrangement transformation and its application in current research in the fields of chemistry and biology.

  9. Oscillating Reactions: Two Analogies

    ERIC Educational Resources Information Center

    Petruševski, Vladimir M.; Stojanovska, Marina I.; Šoptrajanov, Bojan T.

    2007-01-01

    Oscillating chemical reactions are truly spectacular phenomena, and demonstrations are always appreciated by the class. However, explaining such reactions to high school or first-year university students is problematic, because it may seem that no acceptable explanation is possible unless the students have profound knowledge of both physical…

  10. Clock Reaction: Outreach Attraction

    ERIC Educational Resources Information Center

    Carpenter, Yuen-ying; Phillips, Heather A.; Jakubinek, Michael B.

    2010-01-01

    Chemistry students are often introduced to the concept of reaction rates through demonstrations or laboratory activities involving the well-known iodine clock reaction. For example, a laboratory experiment involving thiosulfate as an iodine scavenger is part of the first-year general chemistry laboratory curriculum at Dalhousie University. With…

  11. Chemical burn or reaction

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/000059.htm Chemical burn or reaction To use the sharing features on this page, please enable JavaScript. Chemicals that touch skin can lead to a reaction on the skin, throughout the body, or both. ...

  12. Lipases in lipophilization reactions.

    PubMed

    Villeneuve, Pierre

    2007-01-01

    Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives. PMID:17681737

  13. Enhancing chemical reactions

    DOEpatents

    Morrey, John R.

    1978-01-01

    Methods of enhancing selected chemical reactions. The population of a selected high vibrational energy state of a reactant molecule is increased substantially above its population at thermal equilibrium by directing onto the molecule a beam of radiant energy from a laser having a combination of frequency and intensity selected to pump the selected energy state, and the reaction is carried out with the temperature, pressure, and concentrations of reactants maintained at a combination of values selected to optimize the reaction in preference to thermal degradation by transforming the absorbed energy into translational motion. The reaction temperature is selected to optimize the reaction. Typically a laser and a frequency doubler emit radiant energy at frequencies of .nu. and 2.nu. into an optical dye within an optical cavity capable of being tuned to a wanted frequency .delta. or a parametric oscillator comprising a non-centrosymmetric crystal having two indices of refraction, to emit radiant energy at the frequencies of .nu., 2.nu., and .delta. (and, with a parametric oscillator, also at 2.nu.-.delta.). Each unwanted frequency is filtered out, and each desired frequency is focused to the desired radiation flux within a reaction chamber and is reflected repeatedly through the chamber while reactants are fed into the chamber and reaction products are removed therefrom.

  14. Reactions of oriented molecules.

    PubMed

    Brooks, P R

    1976-07-01

    Beams of oriented molecules have been used to directly study geometrical requirements in chemical reactions. These studies have shown that reactivity is much greater in some orientations than others and demonstrated the existence of steric effects. For some reactions portions of the orientation results are in good accord with traditional views of steric hindrance, but for others it is clear that our chemical intuition needs recalibrating. Indeed, the information gained from simultaneously orienting the reactants and observing the scattering angle of the products may lead to new insights about the detailed mechanism of certain reactions. Further work must be done to extend the scope and detail of the studies described here. More detailed information is needed on the CH(3)I reaction and the CF(3)I reaction. The effects of alkyl groups of various sizes and alkali metals of various sizes are of interest. In addition, reactions where a long-lived complex is formed should be studied to see if orientation is important. Finally, it would be of interest to apply the technique to the sort of reactions that led to our interest in the first place: the S(N)2 displacements in alkyl halides where the fascinating Walden inversion occurs. PMID:17793988

  15. Mechanisms in knockout reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-10-01

    We report on the first detailed study of the mechanisms involved in knockout reactions, via a coincidence measurement of the residue and fast proton in one-proton knockout reactions, using the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results on the reactions ^9Be(^9C,^8B+X)Y and ^9Be(^8B,^7Be+X)Y are presented. They are compared with theoretical predictions for both the diffraction (elastic breakup) and stripping (inelastic breakup) reaction mechanisms, as calculated in the eikonal model. The data shows a clear distinction between the two reaction mechanisms, and the observed respective proportions are very well reproduced by the reaction theory. This agreement supports the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes. In particular, this add considerable support to the use of the eikonal model as a quantitative tool, able, for example, to determine single-particle spectroscopic strengths in rare isotopes.

  16. Hypersensitivity reactions to corticosteroids.

    PubMed

    Vatti, Rani R; Ali, Fatima; Teuber, Suzanne; Chang, Christopher; Gershwin, M Eric

    2014-08-01

    Hypersensitivity reactions to corticosteroids (CS) are rare in the general population, but they are not uncommon in high-risk groups such as patients who receive repeated doses of CS. Hypersensitivity reactions to steroids are broadly divided into two categories: immediate reactions, typically occurring within 1 h of drug administration, and non-immediate reactions, which manifest more than an hour after drug administration. The latter group is more common. We reviewed the literature using the search terms "hypersensitivity to steroids, adverse effects of steroids, steroid allergy, allergic contact dermatitis, corticosteroid side effects, and type I hypersensitivity" to identify studies or clinical reports of steroid hypersensitivity. We discuss the prevalence, mechanism, presentation, evaluation, and therapeutic options in corticosteroid hypersensitivity reactions. There is a paucity of literature on corticosteroid allergy, with most reports being case reports. Most reports involve non-systemic application of corticosteroids. Steroid hypersensitivity has been associated with type I IgE-mediated allergy including anaphylaxis. The overall prevalence of type I steroid hypersensitivity is estimated to be 0.3-0.5%. Allergic contact dermatitis (ACD) is the most commonly reported non-immediate hypersensitivity reaction and usually follows topical CS application. Atopic dermatitis and stasis dermatitis of the lower extremities are risk factors for the development of ACD from topical CS. Patients can also develop hypersensitivity reactions to nasal, inhaled, oral, and parenteral CS. A close and detailed evaluation is required for the clinician to confirm the presence of a true hypersensitivity reaction to the suspected drug and choose the safest alternative. Choosing an alternative CS is not only paramount to the patient's safety but also ameliorates the worry of developing an allergic, and potentially fatal, steroid hypersensitivity reaction. This evaluation becomes

  17. NEUTRONIC REACTION SYSTEM

    DOEpatents

    Wigner, E.P.

    1963-09-01

    A nuclear reactor system is described for breeding fissionable material, including a heat-exchange tank, a high- and a low-pressure chamber therein, heat- exchange tubes connecting these chambers, a solution of U/sup 233/ in heavy water in a reaction container within the tank, a slurry of thorium dioxide in heavy water in a second container surrounding the first container, an inlet conduit including a pump connecting the low pressure chamber to the reaction container, an outlet conduit connecting the high pressure chamber to the reaction container, and means of removing gaseous fission products released in both chambers. (AEC)

  18. Mathematical modeling and application of genetic algorithm to parameter estimation in signal transduction: trafficking and promiscuous coupling of G-protein coupled receptors.

    PubMed

    Modchang, Charin; Triampo, Wannapong; Lenbury, Yongwimon

    2008-05-01

    G-protein-coupled receptors (GPCRs) constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. These receptors play a critical role in signal transduction, and are among the most important pharmacological drug targets. Upon binding of extracellular ligands, these receptor molecules couple to one or several subtypes of G-protein which reside at the intracellular side of the plasma membrane to trigger intracellular signaling events. The question of how GPCRs select and activate a single or multiple G-protein subtype(s) has been the topic of intense investigations. Evidence is also accumulating; however, that certain GPCRs can be internalized via lipid rafts and caveolae. In many cases, the mechanisms responsible for this still remain to be elucidated. In this work, we extend the mathematical model proposed by Chen et al. [Modelling of signalling via G-protein coupled receptors: pathway-dependent agonist potency and efficacy, Bull. Math. Biol. 65 (5) (2003) 933-958] to take into account internalization, recycling, degradation and synthesis of the receptors. In constructing the model, we assume that the receptors can exist in multiple conformational states allowing for a multiple effecter pathways. As data on kinetic reaction rates in the signalling processes measured in reliable in vivo and in vitro experiments is currently limited to a small number of known values. In this paper, we also apply a genetic algorithm (GA) to estimate the parameter values in our model. PMID:18367158

  19. Catalysis in reaction networks.

    PubMed

    Gopalkrishnan, Manoj

    2011-12-01

    We define catalytic networks as chemical reaction networks with an essentially catalytic reaction pathway: one which is "on" in the presence of certain catalysts and "off" in their absence. We show that examples of catalytic networks include synthetic DNA molecular circuits that have been shown to perform signal amplification and molecular logic. Recall that a critical siphon is a subset of the species in a chemical reaction network whose absence is forward invariant and stoichiometrically compatible with a positive point. Our main theorem is that all weakly-reversible networks with critical siphons are catalytic. Consequently, we obtain new proofs for the persistence of atomic event-systems of Adleman et al., and normal networks of Gnacadja. We define autocatalytic networks, and conjecture that a weakly-reversible reaction network has critical siphons if and only if it is autocatalytic. PMID:21503834

  20. Chemisorption And Precipitation Reactions

    EPA Science Inventory

    The transport and bioavailability of chemical components within soils is, in part, controlled by partitioning between solids and solution. General terms used to describe these partitioning reactions include chemisorption and precipitation. Chemisorption is inclusive of the suit...

  1. An Illuminating Reaction.

    ERIC Educational Resources Information Center

    Matthews, Catherine E.

    1996-01-01

    Describes the use of carbide lights as an excellent mechanism for introducing or reviewing many basic chemistry concepts including elements and compounds, endothermic and exothermic reactions, physical and chemical changes, and balancing chemical equations. (JRH)

  2. Reactor for exothermic reactions

    DOEpatents

    Smith, L.A. Jr.; Hearn, D.; Jones, E.M. Jr.

    1993-03-02

    A liquid phase process is described for oligomerization of C[sub 4] and C[sub 5] isoolefins or the etherification thereof with C[sub 1] to C[sub 6] alcohols wherein the reactants are contacted in a reactor with a fixed bed acid cation exchange resin catalyst at an LHSV of 5 to 20, pressure of 0 to 400 psig and temperature of 120 to 300 F. Wherein the improvement is the operation of the reactor at a pressure to maintain the reaction mixture at its boiling point whereby at least a portion but less than all of the reaction mixture is vaporized. By operating at the boiling point and allowing a portion of the reaction mixture to vaporize, the exothermic heat of reaction is dissipated by the formation of more boil up and the temperature in the reactor is controlled.

  3. Iodine Clock Reaction.

    ERIC Educational Resources Information Center

    Mitchell, Richard S.

    1996-01-01

    Describes a combination of solutions that can be used in the study of kinetics using the iodine clock reaction. The combination slows down degradation of the prepared solutions and can be used successfully for several weeks. (JRH)

  4. Untoward penicillin reactions

    PubMed Central

    Guthe, T.; Idsöe, O.; Willcox, R. R.

    1958-01-01

    The literature on untoward reactions following the administration of penicillin is reviewed. These reactions, including a certain number of deaths which have been reported, are of particular interest to health administrations and to WHO in view of the large-scale programmes for controlling the treponematoses which are now under way—programmes affecting millions of people in many parts of the world. The most serious problems are anaphylactic sensitivity phenomena and superinfection or cross-infection with penicillin-resistant organisms, and the reactions involved range in intensity from the mildest to the fatal; the incidence of the latter is estimated at 0.1-0.3 per million injections. The authors point out that with increasing use of penicillin, more persons are likely to become sensitized and the number of reactions can therefore be expected to rise. The best prevention against such an increase is the restriction of the unnecessary use of penicillin. PMID:13596877

  5. Adverse reactions to sulfites

    PubMed Central

    Yang, William H.; Purchase, Emerson C.R.

    1985-01-01

    Sulfites are widely used as preservatives in the food and pharmaceutical industries. In the United States more than 250 cases of sulfite-related adverse reactions, including anaphylactic shock, asthmatic attacks, urticaria and angioedema, nausea, abdominal pain and diarrhea, seizures and death, have been reported, including 6 deaths allegedly associated with restaurant food containing sulfites. In Canada 10 sulfite-related adverse reactions have been documented, and 1 death suspected to be sulfite-related has occurred. The exact mechanism of sulfite-induced reactions is unknown. Practising physicians should be aware of the clinical manifestations of sulfite-related adverse reactions as well as which foods and pharmaceuticals contain sulfites. Cases should be reported to health officials and proper advice given to the victims to prevent further exposure to sulfites. The food industry, including beer and wine manufacturers, and the pharmaceutical industry should consider using alternative preservatives. In the interim, they should list any sulfites in their products. PMID:4052897

  6. Reactor for exothermic reactions

    DOEpatents

    Smith, Jr., Lawrence A.; Hearn, Dennis; Jones, Jr., Edward M.

    1993-01-01

    A liquid phase process for oligomerization of C.sub.4 and C.sub.5 isoolefins or the etherification thereof with C.sub.1 to C.sub.6 alcohols wherein the reactants are contacted in a reactor with a fixed bed acid cation exchange resin catalyst at an LHSV of 5 to 20, pressure of 0 to 400 psig and temperature of 120.degree. to 300.degree. F. Wherein the improvement is the operation of the reactor at a pressure to maintain the reaction mixture at its boiling point whereby at least a portion but less than all of the reaction mixture is vaporized. By operating at the boiling point and allowing a portion of the reaction mixture to vaporize, the exothermic heat of reaction is dissipated by the formation of more boil up and the temperature in the reactor is controlled.

  7. Autocatalysis in reaction networks.

    PubMed

    Deshpande, Abhishek; Gopalkrishnan, Manoj

    2014-10-01

    The persistence conjecture is a long-standing open problem in chemical reaction network theory. It concerns the behavior of solutions to coupled ODE systems that arise from applying mass-action kinetics to a network of chemical reactions. The idea is that if all reactions are reversible in a weak sense, then no species can go extinct. A notion that has been found useful in thinking about persistence is that of "critical siphon." We explore the combinatorics of critical siphons, with a view toward the persistence conjecture. We introduce the notions of "drainable" and "self-replicable" (or autocatalytic) siphons. We show that: Every minimal critical siphon is either drainable or self-replicable; reaction networks without drainable siphons are persistent; and nonautocatalytic weakly reversible networks are persistent. Our results clarify that the difficulties in proving the persistence conjecture are essentially due to competition between drainable and self-replicable siphons. PMID:25245394

  8. Oxygen evolution reaction catalysis

    DOEpatents

    Haber, Joel A.; Jin, Jian; Xiang, Chengxiang; Gregoire, John M.; Jones, Ryan J.; Guevarra, Dan W.; Shinde, Aniketa A.

    2016-09-06

    An Oxygen Evolution Reaction (OER) catalyst includes a metal oxide that includes oxygen, cerium, and one or more second metals. In some instances, the cerium is 10 to 80 molar % of the metals in the metal oxide and/or the catalyst includes two or more second metals. The OER catalyst can be included in or on an electrode. The electrode can be arranged in an oxygen evolution system such that the Oxygen Evolution Reaction occurs at the electrode.

  9. Jets in hadronic reactions

    SciTech Connect

    Paige, F.E.

    1983-01-01

    Recent experimental data on the properties of jets in hadronic reactions are reviewed and compared with theoretical expectations. Jets are clearly established as the dominant process for high E/sub T/ events in hadronic reactions. The cross section and the other properties of these events are in qualitative and even semiquantitative agreement with expectations based on perturbative QCD. However, we can not yet make precise tests of QCD, primarily because there are substantial uncertainties in the theoretical calculations. 45 references. (WHK)

  10. [Cutaneous adverse drug reactions].

    PubMed

    Lebrun-Vignes, B; Valeyrie-Allanore, L

    2015-04-01

    Cutaneous adverse drug reactions (CADR) represent a heterogeneous field including various clinical patterns without specific features suggesting drug causality. Exanthematous eruptions, urticaria and vasculitis are the most common forms of CADR. Fixed eruption is uncommon in western countries. Serious reactions (fatal outcome, sequelae) represent 2% of CADR: bullous reactions (Stevens-Johnson syndrome, toxic epidermal necrolysis), DRESS (drug reaction with eosinophilia and systemic symptoms or drug-induced hypersensitivity syndrome) and acute generalized exanthematous pustulosis (AGEP). These forms must be quickly diagnosed to guide their management. The main risk factors are immunosuppression, autoimmunity and some HLA alleles in bullous reactions and DRESS. Most systemic drugs may induce cutaneous adverse reactions, especially antibiotics, anticonvulsivants, antineoplastic drugs, non-steroidal anti-inflammatory drugs, allopurinol and contrast media. Pathogenesis includes immediate or delayed immunologic mechanism, usually not related to dose, and pharmacologic/toxic mechanism, commonly dose-dependent or time-dependent. In case of immunologic mechanism, allergologic exploration is possible to clarify drug causality, with a variable sensitivity according to the drug and to the CADR type. It includes epicutaneous patch testing, prick test and intradermal test. However, no in vivo or in vitro test can confirm the drug causality. To determine the cause of the eruption, a logical approach based on clinical characteristics, chronologic factors and elimination of differential diagnosis is required, completed with a literature search. A reporting to pharmacovigilance network is essential in case of a serious CADR whatever the suspected drug and in any case if the involved drug is a newly marketed one or unusually related to cutaneous reactions. PMID:25458866

  11. Immediate reaction to clarithromycin.

    PubMed

    Gangemi, S; Ricciardi, L; Fedele, R; Isola, S; Purello-D'Ambrosio, F

    2001-01-01

    We present the case of bronchospastic reaction to clarithromycin had during a drug challenge test. Personal allergic history was negative for respiratory allergies and positive for adverse drug reactions to general and regional anesthesia and to ceftriaxone. After the administration of 1/4 of therapeutic dose of clarithromycin the patient showed dyspnea, cough and bronchospasm in all the lung fields. The positivity of the test was confirmed by the negativity to the administration of placebo. The quickness and the clinical characteristic of the adverse reaction suggest a pathogenic mechanism of immediate-type hypersensitivity. On reviewing the literature we have found no reports of bronchospastic reaction to clarithromycin. Macrolides are a class of antibiotics mainly used in the last years in place of beta-lactams because of a broad spectrum of action and a low allergic power. In fact, there are few reports on allergic reactions to these molecules. Clarithromycin is one of the latest macrolides, characterised by the presence of a 14-carbon-atom lactone ring as erythromycin, active on a wide spectrum of pathogens. PMID:11449533

  12. Find favorable reactions faster

    SciTech Connect

    Yaws, C.L.; Chiang, P.Y. )

    1988-11-01

    Now, equations are given to identify whether the reactions are thermodynamically favorable. The method uses Gibbs free energy of formation for the reactants and products. The equation for any 700 major organic compounds is given as temperature coefficients. Then the reaction can be tested at various temperature levels beyond the standard 298/sup 0/K conditions imposed by many other data tabulations. Data for the water and hydrogen chloride are also included. Gibbs free energy of formation of ideal gas (..delta..G/sub f/, jkoule/g-mol) is calculated from the tabulated coefficients (A, B, C) and the temperature (T, /sup 0/K) using the following equation: (1) ..delta..G/sub f/ = A + BT + CT/sup 2/. Chemical equilibrium for a reaction is associated with the change in Gibbs free energy (..delta..G/sub r/) calculated as follows: (2) ..delta..G/sub r/ = ..delta..G/sub f/, products - ..delta..G/sub f/, reactants. If the change in Gibbs free energy is negative, the thermodynamics for the reaction are favorable. On the other hand, if the change in Gibbs free energy is highly positive, the thermodynamics for the reaction are not favorable and may be feasible only under special circumstances.

  13. Nanoparticle Reactions on Chip

    NASA Astrophysics Data System (ADS)

    Köhler, J. M.; Kirner, Th.; Wagner, J.; Csáki, A.; Möller, R.; Fritzsche, W.

    The handling of heterogenous systems in micro reactors is difficult due to their adhesion and transport behaviour. Therefore, the formation of precipitates and gas bubbles has to be avoided in micro reaction technology, in most cases. But, micro channels and other micro reactors offer interesting possibilities for the control of reaction conditions and transport by diffusion and convection due to the laminar flow caused by small Reynolds numbers. This can be used for the preparation and modification of objects, which are much smaller than the cross section of microchannels. The formation of colloidal solutions and the change of surface states of nano particles are two important tasks for the application of chip reactors in nanoparticle technology. Some concepts for the preparation and reaction of nanoparticles in modular chip reactor arrangements will be discussed.

  14. Hipersensitivity Reactions to Corticosteroids.

    PubMed

    Berbegal, L; DeLeon, F J; Silvestre, J F

    2016-03-01

    Corticosteroids are widely used drugs in the clinical practice, especially by topic application in dermatology. These substances may act as allergens and produce immediate and delayed hypersensitivity reactions. Allergic contact dermatitis is the most frequent presentation of corticosteroid allergy and it should be studied by patch testing in specific units. The corticosteroids included in the Spanish standard battery are good markers but not ideal. Therefore, if those makers are positive, it is useful to apply a specific battery of corticosteroids and the drugs provided by patients. Immediate reactions are relatively rare but potentially severe, and it is important to confirm the sensitization profile and to guide the use of alternative corticosteroids, because they are often necessary in several diseases. In this article we review the main concepts regarding these two types of hypersensitivity reactions in corticosteroid allergy, as well as their approach in the clinical practice. PMID:26621334

  15. Cutaneous reactions to vaccinations.

    PubMed

    Rosenblatt, Adena E; Stein, Sarah L

    2015-01-01

    Vaccinations are important for infectious disease prevention; however, there are adverse effects of vaccines, many of which are cutaneous. Some of these reactions are due to nonspecific inflammation and irritation at the injection site, whereas other reactions are directly related to the live attenuated virus. Rarely, vaccinations have been associated with generalized hypersensitivity reactions, such as erythema multiforme, Stevens-Johnson syndrome, urticaria, acute generalized exanthematous pustulosis, and drug hypersensitivity syndrome. The onset of certain inflammatory dermatologic conditions, such as lichen planus, granuloma annulare, and pemphigoid, were reported to occur shortly after vaccine administration. Allergic contact dermatitis can develop at the injection site, typically due to adjuvant ingredients in the vaccine, such as thimerosal and aluminum. Vaccinations are important to promote development of both individual and herd immunity. Although most vaccinations are considered relatively safe, there may be adverse effects associated with any vaccine. Cutaneous manifestations make up a large portion of the types of reactions associated with vaccines. There are many different reasons for the development of a cutaneous reaction to a vaccination. Some are directly related to the injection of a live attenuated virus, such as varicella or vaccinia (for immunity to smallpox), whereas others cause more nonspecific erythema and swelling at the injection site, as a result of local inflammation or irritation. Vaccinations have also been associated in rare reports with generalized hypersensitivity reactions, such as erythema multiforme, Stevens-Johnson syndrome, urticaria, acute generalized exanthematous pustulosis, and drug hypersensitivity syndrome. There have been case reports associating the administration of a vaccine with the new onset of a dermatologic condition, such as lichen planus, granuloma annulare, and Sweet syndrome. Finally, allergic contact

  16. Velocity pump reaction turbine

    DOEpatents

    House, P.A.

    An expanding hydraulic/two-phase velocity pump reaction turbine including a dual concentric rotor configuration with an inter-rotor annular flow channel in which the inner rotor is mechanically driven by the outer rotor. In another embodiment, the inner rotor is immobilized and provided with gas recovery ports on its outer surface by means of which gas in solution may be recovered. This velocity pump reaction turbine configuration is capable of potential energy conversion efficiencies of up to 70%, and is particularly suited for geothermal applications.

  17. Velocity pump reaction turbine

    DOEpatents

    House, Palmer A.

    1984-01-01

    An expanding hydraulic/two-phase velocity pump reaction turbine including a dual concentric rotor configuration with an inter-rotor annular flow channel in which the inner rotor is mechanically driven by the outer rotor. In another embodiment, the inner rotor is immobilized and provided with gas recovery ports on its outer surface by means of which gas in solution may be recovered. This velocity pump reaction turbine configuration is capable of potential energy conversion efficiencies of up to 70%, and is particularly suited for geothermal applications.

  18. Velocity pump reaction turbine

    DOEpatents

    House, Palmer A.

    1982-01-01

    An expanding hydraulic/two-phase velocity pump reaction turbine including a dual concentric rotor configuration with an inter-rotor annular flow channel in which the inner rotor is mechanically driven by the outer rotor. In another embodiment, the inner rotor is immobilized and provided with gas recovery ports on its outer surface by means of which gas in solution may be recovered. This velocity pump reaction turbine configuration is capable of potential energy conversion efficiencies of up to 70%, and is particularly suited for geothermal applications.

  19. Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

    PubMed Central

    Guzmán, Fanny; Rosas, Fernando; Thomas, M. Carmen; López, Manuel Carlos; González, John Mario; Cuéllar, Adriana; Puerta, Concepción J.

    2016-01-01

    Background TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. Methodology/Principal Findings From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. Conclusion/Significance These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy. PMID:26974162

  20. Quinoprotein-catalysed reactions.

    PubMed Central

    Anthony, C

    1996-01-01

    This review is concerned with the structure and function of the quinoprotein enzymes, sometimes called quinoenzymes. These have prosthetic groups containing quinones, the name thus being analogous to the flavoproteins containing flavin prosthetic groups. Pyrrolo-quinoline quinone (PQQ) is non-covalently attached, whereas tryptophan tryptophylquinone (TTQ), topaquinone (TPQ) and lysine tyrosylquinone (LTQ) are derived from amino acid residues in the backbone of the enzymes. The mechanisms of the quinoproteins are reviewed and related to their recently determined three-dimensional structures. As expected, the quinone structures in the prosthetic groups play important roles in the mechanisms. A second common feature is the presence of a catalytic base (aspartate) at the active site which initiates the reactions by abstracting a proton from the substrate, and it is likely to be involved in multiple reactions in the mechanism. A third common feature of these enzymes is that the first part of the reaction produces a reduced prosthetic group; this part of the mechanism is fairly well understood. This is followed by an oxidative phase involving electron transfer reactions which remain poorly understood. In both types of dehydrogenase (containing PQQ and TTQ), electrons must pass from the reduced prosthetic group to redox centres in a second recipient protein (or protein domain), whereas in amine oxidases (containing TPQ or LTQ), electrons must be transferred to molecular oxygen by way of a redox-active copper ion in the protein. PMID:9003352

  1. Chain Reaction Polymerization.

    ERIC Educational Resources Information Center

    McGrath, James E.

    1981-01-01

    The salient features and importance of chain-reaction polymerization are discussed, including such topics as the thermodynamics of polymerization, free-radical polymerization kinetics, radical polymerization processes, copolymers, and free-radical chain, anionic, cationic, coordination, and ring-opening polymerizations. (JN)

  2. Exocharmic Reactions up Close

    ERIC Educational Resources Information Center

    Ramette, R. W.

    2007-01-01

    The exocharmic reactions that can be observed microscopically are discussed. The students can discover the optimal concentration of an acidic lead nitrate solution, so that a crystal of potassium iodide, nudged to the edge of a drop, results in glinting golden hexagons of lead iodide.

  3. Chemical Reactions at Surfaces

    SciTech Connect

    Michael Henderson and Nancy Ryan Gray

    2010-04-14

    Chemical reactions at surfaces underlie some of the most important processes of today, including catalysis, energy conversion, microelectronics, human health and the environment. Understanding surface chemical reactions at a fundamental level is at the core of the field of surface science. The Gordon Research Conference on Chemical Reactions at Surfaces is one of the premiere meetings in the field. The program this year will cover a broad range of topics, including heterogeneous catalysis and surface chemistry, surfaces in environmental chemistry and energy conversion, reactions at the liquid-solid and liquid-gas interface, electronic materials growth and surface modification, biological interfaces, and electrons and photons at surfaces. An exciting program is planned, with contributions from outstanding speakers and discussion leaders from the international scientific community. The conference provides a dynamic environment with ample time for discussion and interaction. Attendees are encouraged to present posters; the poster sessions are historically well attended and stimulate additional discussions. The conference provides an excellent opportunity for junior researchers (e.g. graduate students or postdocs) to present their work and interact with established leaders in the field.

  4. Radiobiology of tissue reactions.

    PubMed

    Dörr, W

    2015-06-01

    Tissue effects of radiation exposure are observed in virtually all normal tissues, with interactions when several organs are involved. Early reactions occur in turnover tissues, where proliferative impairment results in hypoplasia; late reactions, based on combined parenchymal, vascular, and connective tissue changes, result in loss of function within the exposed volume; consequential late effects develop through interactions between early and late effects in the same organ; and very late effects are dominated by vascular sequelae. Invariably, involvement of the immune system is observed. Importantly, latent times of late effects are inversely dependent on the biologically equieffective dose. Each tissue component and--importantly--each individual symptom/endpoint displays a specific dose-effect relationship. Equieffective doses are modulated by exposure conditions: in particular, dose-rate reduction--down to chronic levels--and dose fractionation impact on late responding tissues, while overall exposure time predominantly affects early (and consequential late) reactions. Consequences of partial organ exposure are related to tissue architecture. In 'tubular' organs (gastrointestinal tract, but also vasculature), punctual exposure affects function in downstream compartments. In 'parallel' organs, such as liver or lungs, only exposure of a significant (organ-dependent) fraction of the total volume results in clinical consequences. Forthcoming studies must address biomarkers of the individual risk for tissue reactions, and strategies to prevent/mitigate tissue effects after exposure. PMID:25816259

  5. Reaction and Response.

    ERIC Educational Resources Information Center

    Armento, Beverly J.; And Others

    1993-01-01

    Provides a reaction by three economic educators to an article by Raymond C. Miller calling for the elimination of economics. Contends that traditional economics does not necessarily lead to the degradation of the environment. Argues that economics should not promote any set of social values. (CFR)

  6. A Principal's Reaction

    ERIC Educational Resources Information Center

    Zaretsky, Lindy

    2004-01-01

    This article presents a principal's reaction to Catherine Marshall and Michael Ward's article on research on social justice and training for leadership. The author applauds Marshall and Ward's efforts to address what is undoubtedly among the most fundamentally important issues facing principals today. Marshall and Ward illuminate the importance of…

  7. Reaction product imaging

    SciTech Connect

    Chandler, D.W.

    1993-12-01

    Over the past few years the author has investigated the photochemistry of small molecules using the photofragment imaging technique. Bond energies, spectroscopy of radicals, dissociation dynamics and branching ratios are examples of information obtained by this technique. Along with extending the technique to the study of bimolecular reactions, efforts to make the technique as quantitative as possible have been the focus of the research effort. To this end, the author has measured the bond energy of the C-H bond in acetylene, branching ratios in the dissociation of HI, the energetics of CH{sub 3}Br, CD{sub 3}Br, C{sub 2}H{sub 5}Br and C{sub 2}H{sub 5}OBr dissociation, and the alignment of the CD{sub 3} fragment from CD{sub 3}I photolysis. In an effort to extend the technique to bimolecular reactions the author has studied the reaction of H with HI and the isotopic exchange reaction between H and D{sub 2}.

  8. Reactions to Others' Intimacy.

    ERIC Educational Resources Information Center

    Neufeldt, David E.; Olinger, Evanelle J.

    Research using behavioral measures has indicated that men react less positively to the touch of a same sex individual than women, that both men and women react more positively to the touch of an opposite sex individual than to the touch of a same sex individual, and that men and women do not differ in their reactions to opposite sex touch. This…

  9. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  10. A Superintendent's Reaction

    ERIC Educational Resources Information Center

    Lytle, James H.

    2004-01-01

    This article presents a superintendent's reaction to Catherine Marshall and Michael Ward's article on research on social justice and training for leadership. The author states that there is a problem with Marshall and Ward's article which begins with the title, particularly with the word "training." The author contends that there is a significant…

  11. Enantioselective Vinylogous Organocascade Reactions.

    PubMed

    Hepburn, Hamish B; Dell'Amico, Luca; Melchiorre, Paolo

    2016-08-01

    Cascade reactions are powerful tools for rapidly assembling complex molecular architectures from readily available starting materials in a single synthetic operation. Their marriage with asymmetric organocatalysis has led to the development of novel techniques, which are now recognized as reliable strategies for the one-pot enantioselective synthesis of stereochemically dense molecules. In recent years, even more complex synthetic challenges have been addressed by applying the principle of vinylogy to the realm of organocascade catalysis. The key to the success of vinylogous organocascade reactions is the unique ability of the chiral organocatalyst to transfer reactivity to a distal position without losing control on the stereo-determining events. This approach has greatly expanded the synthetic horizons of the field by providing the possibility of forging multiple stereocenters in remote positions from the catalyst's point of action with high selectivity, while simultaneously constructing multiple new bonds. This article critically describes the developments achieved in the field of enantioselective vinylogous organocascade reactions, charting the ideas, the conceptual advances, and the milestone reactions that have been essential for reaching highly practical levels of synthetic efficiency. PMID:27256039

  12. Reaction Formulation: A Bibliography.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    Reaction formation was studied by Sigmund Freud. This defense mechanism may be related to repression, substitution, reversal, and compensation (or over-compensation). Alfred Adler considered compensation a basic process in his individual psychology. Anna Freud discussed some defense mechanisms, and Bibring, Dwyer, Huntington, and Valenstein…

  13. Azlactone Reaction Developments.

    PubMed

    de Castro, Pedro P; Carpanez, Arthur G; Amarante, Giovanni W

    2016-07-18

    Azlactones (also known as oxazolones) are heterocycles usually employed in the stereoselective synthesis of α,α-amino acids, heterocycles and natural products. The versatility of the azlactone scaffold arises from the numerous reactive sites, allowing its application in a diversity of transformations. This review aims to cover classical and recent applications of oxazolones, especially those involving stereoselective processes. After a short introduction on their structures and intrinsic reactivities, dynamic kinetic resolution (DKR) processes as well as reactions involving stereoselective formation of a new σ C-C bond, such as alkylation/allylation/arylation, aldol, ene, Michael and Mannich reactions will be exposed. Additionally, cycloadditions, Steglich rearrangement and sulfenylation reactions will also be discussed. Recent developments of the well-known Erlenmeyer azlactones will be described. For the most examples, the proposed mechanism, activation modes and/or key reaction intermediates will be exposed to rationalize both the final product and the observed stereochemistry. Finally, this review gives an overview of the synthetic utility of oxazolones. PMID:27245128

  14. Photoneutron reactions in astrophysics

    SciTech Connect

    Varlamov, V. V. Ishkhanov, B. S.; Orlin, V. N.; Peskov, N. N.; Stopani, K. A.

    2014-12-15

    Among key problems in nuclear astrophysics, that of obtaining deeper insight into the mechanism of synthesis of chemical elements is of paramount importance. The majority of heavy elements existing in nature are produced in stars via radiative neutron capture in so-called s- and r processes, which are, respectively, slow and fast, in relation to competing β{sup −}-decay processes. At the same time, we know 35 neutron-deficient so-called bypassed p-nuclei that lie between {sup 74}Se and {sup 196}Hg and which cannot originate from the aforementioned s- and r-processes. Their production is possible in (γ, n), (γ, p), or (γ, α) photonuclear reactions. In view of this, data on photoneutron reactions play an important role in predicting and describing processes leading to the production of p-nuclei. Interest in determining cross sections for photoneutron reactions in the threshold energy region, which is of particular importance for astrophysics, has grown substantially in recent years. The use of modern sources of quasimonoenergetic photons obtained in processes of inverse Compton laser-radiation scattering on relativistic electronsmakes it possible to reveal rather interesting special features of respective cross sections, manifestations of pygmy E1 and M1 resonances, or the production of nuclei in isomeric states, on one hand, and to revisit the problem of systematic discrepancies between data on reaction cross sections from experiments of different types, on the other hand. Data obtained on the basis of our new experimental-theoretical approach to evaluating cross sections for partial photoneutron reactions are invoked in considering these problems.

  15. Water-gas shift reaction

    SciTech Connect

    Newsome, D.S.

    1980-01-01

    A review covers the industrial applications of the water-gas shift reaction in hydrogen manufacturing, removing CO from ammonia synthesis feeds, and detoxifying town gas; and the catalyst characteristics, reaction kinetics, and reaction mechanisms of the water-gas shift reactions catalyzed by iron-based, copper-based, or sulfided cobalt-molybdenum catalysts.

  16. Inorganic Reaction Mechanisms. Part I

    ERIC Educational Resources Information Center

    Cooke, D. O.

    1976-01-01

    Provides a collection of data on the mechanistic aspects of inorganic chemical reactions. Wherever possible includes procedures for classroom demonstration or student project work. The material covered includes gas phase reactions, reactions in solution, mechanisms of electron transfer, the reaction between iron III and iodine, and hydrolysis. (GS)

  17. What Is a Reaction Rate?

    ERIC Educational Resources Information Center

    Schmitz, Guy

    2005-01-01

    The definition of reaction rate is derived and demonstrations are made for the care to be taken while using the term. Reaction rate can be in terms of a reaction property, the extent of reaction and thus it is possible to give a definition applicable in open and closed systems.

  18. Dearomatization through Halofunctionalization Reactions.

    PubMed

    Liang, Xiao-Wei; Zheng, Chao; You, Shu-Li

    2016-08-16

    Recent advances in dearomatization through halofunctionalization reactions are summarized in this Minireview. Two general categories of strategies are currently employed in this field. On one hand, the reaction can be initiated with electrophilic halogenation at an alkyne or alkene moiety. The resulting halonium ion intermediate is then captured by a pendant aromatic ring at the ipso position, affording the dearomatization product. On the other hand, electrophilic halogenation can directly take place at a substituted arene, and the final dearomatization product is furnished by deprotonation or intramolecular nucleophilic trap. Highly enantioselective variants have been realized in the latter case by organocatalysis or transition metal catalysis. By applying these methods, various valuable halogenated polycyclic molecular architectures have been obtained from readily available starting materials. PMID:27377184

  19. Reaction chemistry of cerium

    SciTech Connect

    1997-01-01

    It is truly ironic that a synthetic organic chemist likely has far greater knowledge of the reaction chemistry of cerium(IV) than an inorganic colleague. Cerium(IV) reagents have long since been employed as oxidants in effecting a wide variety of organic transformations. Conversely, prior to the late 1980s, the number of well characterized cerium(IV) complexes did not extend past a handful of known species. Though in many other areas, interest in the molecular chemistry of the 4f-elements has undergone an explosive growth over the last twenty years, the chemistry of cerium(IV) has for the most part been overlooked. This report describes reactions of cerium complexes and structure.

  20. Magnetically suspended reaction wheels

    NASA Technical Reports Server (NTRS)

    Sabnis, A. V.; Stocking, G. L.; Dendy, J. B.

    1975-01-01

    Magnetic suspensions offer several advantages over conventional bearings, arising because of the contactless nature of the load support. In application to spacecraft reaction wheels, the advantages are low drag torque, wearfree, unlubricated, vacuum-compatible operation, and unlimited life. By the provision of redundancy in the control electronics, single-point failures are eliminated. The rational for selection of a passive radial, active axial, dc magnetic suspension is presented, and the relative merits of 3-loop and single-loop magnetic suspensions are discussed. The design of a .678 N-m-sec (.5 ft-lb-sec) reaction wheel using the single loop magnetic suspension was developed; the design compares favorably with current ball bearing wheels in terms of weight and power.

  1. Polymerase chain displacement reaction.

    PubMed

    Harris, Claire L; Sanchez-Vargas, Irma J; Olson, Ken E; Alphey, Luke; Fu, Guoliang

    2013-02-01

    Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics. PMID:23384180

  2. Photochemical reaction dynamics

    SciTech Connect

    Moore, B.C.

    1993-12-01

    The purpose of the program is to develop a fundamental understanding of unimolecular and bimolecular reaction dynamics with application in combustion and energy systems. The energy dependence in ketene isomerization, ketene dissociation dynamics, and carbonyl substitution on organometallic rhodium complexes in liquid xenon have been studied. Future studies concerning unimolecular processes in ketene as well as energy transfer and kinetic studies of methylene radicals are discussed.

  3. Nonhemolytic, noninfectious transfusion reactions.

    PubMed

    Barton, J C

    1981-04-01

    The delivery of optimal transfusion therapy requires that the physician first have a thorough understanding of his patient's disease and prior transfusion history. Sometimes the need for blood product administration is more apparent than real. In the selection of necessary therapy, particular blood components, their volumes, and the timing of their administration should be carefully planned. The transfusion of whole blood, particularly as single-unit transfusions, is rarely indicated. Often forgotten, autotransfusion represents a means whereby many subjects who have repeated, unusual, or severe reactions may receive safe treatment. An appreciation of the frequency and manifestations of transfusion-related problems permits effective treatment of ongoing reactions. The prophylactic measures which should be taken against future reactions in most patients are specific, and are the responsibility of the clinician, based upon his bedside observations and laboratory studies. Problems should be discussed with either a hematologist, pathologist, or blood banking expert without hesitation. These guidelines help conserve a precious resource and assure that safe, effective, and economical transfusion therapy is available for all patients in need. PMID:6164098

  4. On-surface reactions.

    PubMed

    Lindner, Robert; Kühnle, Angelika

    2015-06-01

    On-surface synthesis constitutes a rapidly growing field of research due to its promising application for creating stable molecular structures on surfaces. While self-assembled structures rely on reversible interactions, on-surface synthesis provides the potential for creating long-term stable structures with well-controlled properties, for example superior electron transport for future molecular electronic devices. On-surface synthesis holds the promise for preparing insoluble compounds that cannot be produced in solution. Another highly exciting aspect of on-surface synthesis is the chance to discover new reaction pathways due to the two-dimensional confinement of the reaction educts. In this review, we discuss the current state-of-the-art and classify the reactions that have been successfully performed so far. Special emphasis is put on electrically insulating surfaces, as these substrates pose particular challenges for on-surface synthesis while at the same time bearing high potential for future use, for example, in molecular electronics. PMID:25965579

  5. ADVERSE CUTANEOUS DRUG REACTION

    PubMed Central

    Nayak, Surajit; Acharjya, Basanti

    2008-01-01

    In everyday clinical practice, almost all physicians come across many instances of suspected adverse cutaneous drug reactions (ACDR) in different forms. Although such cutaneous reactions are common, comprehensive information regarding their incidence, severity and ultimate health effects are often not available as many cases go unreported. It is also a fact that in the present world, almost everyday a new drug enters market; therefore, a chance of a new drug reaction manifesting somewhere in some form in any corner of world is unknown or unreported. Although many a times, presentation is too trivial and benign, the early identification of the condition and identifying the culprit drug and omit it at earliest holds the keystone in management and prevention of a more severe drug rash. Therefore, not only the dermatologists, but all practicing physicians should be familiar with these conditions to diagnose them early and to be prepared to handle them adequately. However, we all know it is most challenging and practically difficult when patient is on multiple medicines because of myriad clinical symptoms, poorly understood multiple mechanisms of drug-host interaction, relative paucity of laboratory testing that is available for any definitive and confirmatory drug-specific testing. Therefore, in practice, the diagnosis of ACDR is purely based on clinical judgment. In this discussion, we will be primarily focusing on pathomechanism and approach to reach a diagnosis, which is the vital pillar to manage any case of ACDR. PMID:19967009

  6. Chemical Reactions in DSMC

    SciTech Connect

    Bird, G. A.

    2011-05-20

    DSMC simulations of chemically reacting gas flows have generally employed procedures that convert the macroscopic chemical rate equations to reaction cross-sections at the microscopic level. They therefore depend on the availability of experimental data that has been fitted to equations of the Arrhenius form. This paper presents a physical model for dissociation and recombination reactions and a phenomenological model for exchange and chain reactions. These are based on the vibrational states of the colliding molecules and do not require any experimentally-based data. The simplicity of the models allows the corresponding rate equations to be written down and, while these are not required for the implementation of the models, they facilitate their validation. The model is applied to a typical hypersonic atmospheric entry problem and the results are compared with the corresponding results from the traditional method. It is also used to investigate both spontaneous and forced ignition as well as the structure of a deflagration wave in an oxygen-hydrogen mixture.

  7. Chemical Reactions in DSMC

    NASA Astrophysics Data System (ADS)

    Bird, G. A.

    2011-05-01

    DSMC simulations of chemically reacting gas flows have generally employed procedures that convert the macroscopic chemical rate equations to reaction cross-sections at the microscopic level. They therefore depend on the availability of experimental data that has been fitted to equations of the Arrhenius form. This paper presents a physical model for dissociation and recombination reactions and a phenomenological model for exchange and chain reactions. These are based on the vibrational states of the colliding molecules and do not require any experimentally-based data. The simplicity of the models allows the corresponding rate equations to be written down and, while these are not required for the implementation of the models, they facilitate their validation. The model is applied to a typical hypersonic atmospheric entry problem and the results are compared with the corresponding results from the traditional method. It is also used to investigate both spontaneous and forced ignition as well as the structure of a deflagration wave in an oxygen-hydrogen mixture.

  8. Adolescent Formula Literature and Its Promiscuous Progeny.

    ERIC Educational Resources Information Center

    Stanek, Lou Willett

    This paper discusses the history and effect of popular culture generally and of the adolescent formula novel specifically. Seven primary characteristics of art as popular culture are that the work is accessible, easy to understand, conventional in form, not shocking in content, expressive of common and appropriate values, relative to some element…

  9. Black widow pulsars: the price of promiscuity

    NASA Astrophysics Data System (ADS)

    King, A. R.; Davies, M. B.; Beer, M. E.

    2003-10-01

    The incidence of evaporating `black widow' pulsars (BWPs) among all millisecond pulsars is far higher in globular clusters than in the field. This implies a special formation mechanism for them in clusters. Cluster millisecond pulsars in wide binaries with white dwarf companions exchange them for turnoff-mass stars. These new companions eventually overflow their Roche lobes because of encounters and tides. The millisecond pulsars eject the overflowing gas from the binary, giving mass loss on the binary evolution time-scale. The systems are only observable as BWPs at epochs where this evolution is slow, making the mass loss transparent and the lifetime long. This explains why observed BWPs have low-mass companions. We suggest that at least some field BWPs were ejected from globular clusters or entered the field population when the cluster itself was disrupted.

  10. Was Jane Addams a Promiscuous Pragmatist?

    ERIC Educational Resources Information Center

    Atkinson, Becky

    2013-01-01

    Contemporary pragmatist and feminist scholars have proposed the possibilities for "changing the theoretical analyses and concrete practices" of both feminism and classical American pragmatism offered by its recuperation through feminism. Particularly, scholarship on Jane Addams has reached back to retrieve her activism, ethics, and…

  11. Procedures for Decomposing a Redox Reaction into Half-Reaction

    ERIC Educational Resources Information Center

    Fishtik, Ilie; Berka, Ladislav H.

    2005-01-01

    A simple algorithm for a complete enumeration of the possible ways a redox reaction (RR) might be uniquely decomposed into half-reactions (HRs) using the response reactions (RERs) formalism is presented. A complete enumeration of the possible ways a RR may be decomposed into HRs is equivalent to a complete enumeration of stoichiometrically…

  12. Reaction Extrema: Extent of Reaction in General Chemistry

    ERIC Educational Resources Information Center

    Vandezande, Jonathon E.; Vander Griend, Douglas A.; DeKock, Roger L.

    2013-01-01

    Nearly 100 years ago de Donder introduced the term "extent of reaction", ?. We build on that work by defining the concept of reagent extrema for an arbitrary chemical reaction, aA + bB [reversible reaction] yY + zZ. The central equation is ?^[subscript i] = -n[subscript i,0]/?[subscript i]. The symbol ?^[subscript i] represents the…

  13. Insect bite reactions.

    PubMed

    Singh, Sanjay; Mann, Baldeep Kaur

    2013-01-01

    Insects are a class of living creatures within the arthropods. Insect bite reactions are commonly seen in clinical practice. The present review touches upon the medically important insects and their places in the classification, the sparse literature on the epidemiology of insect bites in India, and different variables influencing the susceptibility of an individual to insect bites. Clinical features of mosquito bites, hypersensitivity to mosquito bites Epstein-Barr virus NK (HMB-EBV-NK) disease, eruptive pseudoangiomatosis, Skeeter syndrome, papular pruritic eruption of HIV/AIDS, and clinical features produced by bed bugs, Mexican chicken bugs, assassin bugs, kissing bugs, fleas, black flies, Blandford flies, louse flies, tsetse flies, midges, and thrips are discussed. Brief account is presented of the immunogenic components of mosquito and bed bug saliva. Papular urticaria is discussed including its epidemiology, the 5 stages of skin reaction, the SCRATCH principle as an aid in diagnosis, and the recent evidence supporting participation of types I, III, and IV hypersensitivity reactions in its causation is summarized. Recent developments in the treatment of pediculosis capitis including spinosad 0.9% suspension, benzyl alcohol 5% lotion, dimethicone 4% lotion, isopropyl myristate 50% rinse, and other suffocants are discussed within the context of evidence derived from randomized controlled trials and key findings of a recent systematic review. We also touch upon a non-chemical treatment of head lice and the ineffectiveness of egg-loosening products. Knockdown resistance (kdr) as the genetic mechanism making the lice nerves insensitive to permethrin is discussed along with the surprising contrary clinical evidence from Europe about efficacy of permethrin in children with head lice carrying kdr-like gene. The review also presents a brief account of insects as vectors of diseases and ends with discussion of prevention of insect bites and some serious adverse effects

  14. Copper mediated carbometalation reactions.

    PubMed

    Müller, D S; Marek, I

    2016-08-01

    Since the first discovery of carbocupration of alkynes in the 1970s a tremendous amount of research has been carried out in this field. The exceptionally high selectivities obtained attribute to the great synthetic value of carbocupration reactions. This tutorial review will present the most important features of carbocupration of alkynes and highlight the most relevant reviews. Then a comprehensive review of copper mediated carbometalation of cyclopropenes will follow. The latter method has received much attention over the last decade as it allows the highly selective construction of poly-substituted cyclopropanes which can be transformed into acyclic derivatives bearing one or multiple tertiary or quaternary carbon stereocenters. PMID:26808300

  15. Electronegativity and redox reactions.

    PubMed

    Miranda-Quintana, Ramón Alain; Martínez González, Marco; Ayers, Paul W

    2016-08-10

    Using the maximum hardness principle, we show that the oxidation potential of a molecule increases as its electronegativity increases and also increases as its electronegativity in its oxidized state increases. This insight can be used to construct a linear free energy relation for the oxidation potential, which we train on a set of 31 organic redox couples and test on a set of 10 different redox reactions. Better results are obtained when the electronegativity of the oxidized/reduced reagents are adjusted to account for the reagents' interaction with their chemical environment. PMID:27451962

  16. Organic chemistry: Reactions triggered electrically

    NASA Astrophysics Data System (ADS)

    Xiang, Limin; Tao, N. J.

    2016-03-01

    Single-molecule experiments have revealed that chemical reactions can be controlled using electric fields -- and that the reaction rate is sensitive to both the direction and the strength of the applied field. See Letter p.88

  17. Positive reaction to allergen (image)

    MedlinePlus

    Allergic reaction is a sensitivity to a specific substance, called an allergen, that is contacted through the skin, inhaled into the lungs, swallowed or injected. The body's reaction to an allergen can be mild, such as ...

  18. Hydrazine decomposition and other reactions

    NASA Technical Reports Server (NTRS)

    Armstrong, Warren E. (Inventor); La France, Donald S. (Inventor); Voge, Hervey H. (Inventor)

    1978-01-01

    This invention relates to the catalytic decomposition of hydrazine, catalysts useful for this decomposition and other reactions, and to reactions in hydrogen atmospheres generally using carbon-containing catalysts.

  19. Demonstration of the Fenton Reaction

    ERIC Educational Resources Information Center

    Luehrs, Dean C.; Roher, Alex E.

    2007-01-01

    The study demonstrates the Fenton reaction, which is carried out using the Fenton reagent that is used for groundwater and soil remediation. The Fenton reaction can be implicated in DNA damage, Alzheimer's disease, cardiovascular disease and ageing in general.

  20. Allergic reactions to medication (image)

    MedlinePlus

    A true allergy to a medication is different than a simple adverse reaction to the drug. The allergic reaction occurs when the immune system, having been exposed to the drug before, creates antibodies to ...

  1. ''Subthreshold'' reactions involving nuclear fission

    SciTech Connect

    Goldhaber, M.; Shrock, R.

    2001-02-01

    We analyze reactions of several types that are naively below threshold but can proceed because of the release of binding energy from nuclear fission and occasionally the formation of Coulombic bound states. These reactions include (i) photofission with pion production and (ii) charged current neutrino-nucleus reactions that lead to fission and/or formation of a Coulomb bound state of a {mu}{sup -} with the nucleus of a fission fragment. We comment on the possible experimental observation of these reactions.

  2. The Vitamin C Clock Reaction

    NASA Astrophysics Data System (ADS)

    Wright, Stephen W.

    2002-01-01

    An iodine clock reaction that gives a colorless to black result similar to that of the familiar Landolt iodate-bisulfite clock reaction is described. The vitamin C clock reaction uses chemicals that are readily available on the retail market: vitamin C, tincture of iodine, 3% hydrogen peroxide, and laundry starch. Orange juice may be used as the vitamin C source to give an orange to black reaction.

  3. More on Chemical Reaction Balancing.

    ERIC Educational Resources Information Center

    Swinehart, D. F.

    1985-01-01

    A previous article stated that only the matrix method was powerful enough to balance a particular chemical equation. Shows how this equation can be balanced without using the matrix method. The approach taken involves writing partial mathematical reactions and redox half-reactions, and combining them to yield the final balanced reaction. (JN)

  4. The Vitamin C Clock Reaction.

    ERIC Educational Resources Information Center

    Wright, Stephen W.

    2002-01-01

    Describes an iodine clock reaction that produces an effect similar to the Landolt clock reaction. This reaction uses supermarket chemicals and avoids iodate, bisulfite, and mercury compounds. Ascorbic acid and tincture of iodine are the main reactants with alternate procedures provided for vitamin C tablets and orange juice. (DDR)

  5. Development of detonation reaction engine

    NASA Technical Reports Server (NTRS)

    Lange, O. H.; Stein, R. J.; Tubbs, H. E.

    1968-01-01

    Reaction engine operates on the principle of a controlled condensed detonation. In this engine the gas products that are expelled from the engine to produce thrust are generated by the condensed detonation reaction. The engine is constructed of two basic sections consisting of a detonation wave generator section and a condensed detonation reaction section.

  6. Mass Transfer with Chemical Reaction.

    ERIC Educational Resources Information Center

    DeCoursey, W. J.

    1987-01-01

    Describes the organization of a graduate course dealing with mass transfer, particularly as it relates to chemical reactions. Discusses the course outline, including mathematics models of mass transfer, enhancement of mass transfer rates by homogeneous chemical reaction, and gas-liquid systems with chemical reaction. (TW)

  7. A reaction path study of the catalysis and inhibition of the Bacillus anthracis CapD γ-glutamyl transpeptidase.

    PubMed

    Khavrutskii, Ilja V; Legler, Patricia M; Friedlander, Arthur M; Wallqvist, Anders

    2014-11-11

    The CapD enzyme of Bacillus anthracis is a γ-glutamyl transpeptidase from the N-terminal nucleophile hydrolase superfamily that covalently anchors the poly-γ-D-glutamic acid (pDGA) capsule to the peptidoglycan. The capsule hinders phagocytosis of B. anthracis by host cells and is essential for virulence. The role CapD plays in capsule anchoring and remodeling makes the enzyme a promising target for anthrax medical countermeasures. Although the structure of CapD is known, and a covalent inhibitor, capsidin, has been identified, the mechanisms of CapD catalysis and inhibition are poorly understood. Here, we used a computational approach to map out the reaction steps involved in CapD catalysis and inhibition. We found that the rate-limiting step of either CapD catalysis or inhibition was a concerted asynchronous formation of the tetrahedral intermediate with a barrier of 22-23 kcal/mol. However, the mechanisms of these reactions differed for the two amides. The formation of the tetrahedral intermediate with pDGA was substrate-assisted with two proton transfers. In contrast, capsidin formed the tetrahedral intermediate in a conventional way with one proton transfer. Interestingly, capsidin coupled a conformational change in the catalytic residue of the tetrahedral intermediate to stretching of the scissile amide bond. Furthermore, capsidin took advantage of iminol-amide tautomerism of its diacetamide moiety to convert the tetrahedral intermediate to the acetylated CapD. As evidence of the promiscuous nature of CapD, the enzyme cleaved the amide bond of capsidin by attacking it on the opposite side compared to pDGA. PMID:25334088

  8. Hydrocracking reactions and catalysis

    SciTech Connect

    Dolbear, G.E.

    1995-12-31

    Hydrocracking processes convert aromatic gas oils into high quality gasoline, diesel, and turbine stocks. In doing this, they saturate aromatic rings, crack naphthenes and paraffins, and saturate olefins formed during cracking. The organic chemistry of these steps is well known. Catalysts for hydrocracking contain components for both the hydrogenation and cracking reactions. Hydrogenation activity is provided by Pd or promoted molybdenum or tungsten sulfides. Cracking takes place on strong acid sites in zeolites or amorphous silica aluminas. Specialty catalysts including narrow pore zeolites are used in dewaxing tube oil stocks. Basic nitrogen compounds such as quinoline can poison the acid sites. They are usually removed in a pretreating step, typically with a nickel/molybdenum sulfide catalyst that also removes sulfur.

  9. Laser induced nuclear reactions

    SciTech Connect

    Ledingham, Ken; McCanny, Tom; Graham, Paul; Fang Xiao; Singhal, Ravi; Magill, Joe; Creswell, Alan; Sanderson, David; Allott, Ric; Neely, David; Norreys, Peter; Santala, Marko; Zepf, Matthew; Watts, Ian; Clark, Eugene; Krushelnick, Karl; Tatarakis, Michael; Dangor, Bucker; Machecek, Antonin; Wark, Justin

    1998-12-16

    Dramatic improvements in laser technology since 1984 have revolutionised high power laser technology. Application of chirped-pulse amplification techniques has resulted in laser intensities in excess of 10{sup 19} W/cm{sup 2}. In the mid to late eighties, C. K. Rhodes and K. Boyer discussed the possibility of shining laser light of this intensity onto solid surfaces and to cause nuclear transitions. In particular, irradiation of a uranium target could induce electro- and photofission in the focal region of the laser. In this paper it is shown that {mu}Ci of {sup 62}Cu can be generated via the ({gamma},n) reaction by a laser with an intensity of about 10{sup 19} Wcm{sup -2}.

  10. ISMP Adverse Drug Reactions

    PubMed Central

    2013-01-01

    The purpose of this feature is to heighten awareness of specific adverse drug reactions (ADRs), discuss methods of prevention, and promote reporting of ADRs to the US Food and Drug Administration’s (FDA’s) MedWatch program (800-FDA-1088). If you have reported an interesting, preventable ADR to MedWatch, please consider sharing the account with our readers. Write to Dr. Mancano at ISMP, 200 Lakeside Drive, Suite 200, Horsham, PA 19044 (phone: 215-707-4936; e-mail: mmancano@temple.edu). Your report will be published anonymously unless otherwise requested. This feature is provided by the Institute for Safe Medication Practices (ISMP) in cooperation with the FDA’s MedWatch program and Temple University School of Pharmacy. ISMP is an FDA MedWatch partner. PMID:24421544

  11. Prebiotic photosynthetic reactions.

    PubMed

    Chittenden, G J; Schwartz, A W

    1981-01-01

    Historically, numerous attempts have been made to mimic - by means of inorganic model reactions - the photosynthetic fixation of CO2 by green plants. The literature in this field is strewn with claims and counter-claims. Two factors have led us to reexamine this subject: firstly; doubts concerning the highly reducing model for the atmosphere of the primitive Earth and secondly; recent results which demonstrate that photoreductive fixation is feasable on a suitable catalytic surface, for both CO2 and N2. The latter observation is of particular interest due to the well-known susceptibility of NH3 to photolytic destruction. Our review of the literature leads us to suggest that similar processes would have been plausible for the primitive Earth and could have been prebiotic precursors to an early development of CO2-fixing autotrophs. PMID:6791723

  12. Subdiffusion-reaction processes with A→B reactions versus subdiffusion-reaction processes with A+B→B reactions.

    PubMed

    Kosztołowicz, Tadeusz; Lewandowska, Katarzyna D

    2014-09-01

    We consider the subdiffusion-reaction process with reactions of a type A+B→B (in which particles A are assumed to be mobile, whereas B are assumed to be static) in comparison to the subdiffusion-reaction process with A→B reactions which was studied by Sokolov, Schmidt, and Sagués [Phys. Rev. E 73, 031102 (2006)]. In both processes a rule that reactions can only occur between particles which continue to exist is taken into account. Although in both processes a probability of the vanishing of particle A due to a reaction is independent of both time and space variables (assuming that in the system with the A+B→B reactions, particles B are distributed homogeneously), we show that subdiffusion-reaction equations describing these processes as well as their Green's functions are qualitatively different. The reason for this difference is as follows. In the case of the former reaction, particles A and B have to meet with some probability before the reaction occurs in contradiction with the case of the latter reaction. For the subdiffusion process with the A+B→B reactions we consider three models which differ in some details concerning a description of the reactions. We base the method considered in this paper on a random walk model in a system with both discrete time and discrete space variables. Then the system with discrete variables is transformed into a system with both continuous time and continuous space variables. Such a method seems to be convenient in analyzing subdiffusion-reaction processes with partially absorbing or partially reflecting walls. The reason is that within this method we can determine Green's functions without a necessity of solving a fractional differential subdiffusion-reaction equation with boundary conditions at the walls. As an example, we use the model to find the Green's functions for a subdiffusive reaction system (with the reactions mentioned above), which is bounded by a partially absorbing wall. This example shows how the model

  13. Nuclear Reactions on Unstable Nuclei and the Surrogate Reaction Technique

    SciTech Connect

    Escher, J

    2004-03-01

    Determining reaction cross sections on short-lived nuclear species is a major challenge for nuclear physics and nuclear astrophysics. Many of these nuclei are too difficult to produce with currently available experimental techniques or too short-lived to serve as targets in present-day set-ups. Some nuclear reactions will remain immeasurable even at upcoming and planned radioactive beam facilities. It is therefore important to explore alternative methods for determining reaction cross sections on unstable nuclei.

  14. Communication: Resonance reaction in diffusion-influenced bimolecular reactions.

    PubMed

    Kolb, Jakob J; Angioletti-Uberti, Stefano; Dzubiella, Joachim

    2016-02-28

    We investigate the influence of a stochastically fluctuating step-barrier potential on bimolecular reaction rates by exact analytical theory and stochastic simulations. We demonstrate that the system exhibits a new "resonant reaction" behavior with rate enhancement if an appropriately defined fluctuation decay length is of the order of the system size. Importantly, we find that in the proximity of resonance, the standard reciprocal additivity law for diffusion and surface reaction rates is violated due to the dynamical coupling of multiple kinetic processes. Together, these findings may have important repercussions on the correct interpretation of various kinetic reaction problems in complex systems, as, e.g., in biomolecular association or catalysis. PMID:26931674

  15. Communication: Resonance reaction in diffusion-influenced bimolecular reactions

    NASA Astrophysics Data System (ADS)

    Kolb, Jakob J.; Angioletti-Uberti, Stefano; Dzubiella, Joachim

    2016-02-01

    We investigate the influence of a stochastically fluctuating step-barrier potential on bimolecular reaction rates by exact analytical theory and stochastic simulations. We demonstrate that the system exhibits a new "resonant reaction" behavior with rate enhancement if an appropriately defined fluctuation decay length is of the order of the system size. Importantly, we find that in the proximity of resonance, the standard reciprocal additivity law for diffusion and surface reaction rates is violated due to the dynamical coupling of multiple kinetic processes. Together, these findings may have important repercussions on the correct interpretation of various kinetic reaction problems in complex systems, as, e.g., in biomolecular association or catalysis.

  16. Two chamber reaction furnace

    DOEpatents

    Blaugher, R.D.

    1998-05-05

    A vertical two chamber reaction furnace is described. The furnace comprises a lower chamber having an independently operable first heating means for heating the lower chamber and a gas inlet means for admitting a gas to create an ambient atmosphere, and an upper chamber disposed above the lower chamber and having an independently operable second heating means for heating the upper chamber. Disposed between the lower chamber and the upper chamber is a vapor permeable diffusion partition. The upper chamber has a conveyor means for conveying a reactant there through. Of particular importance is the thallinating of long-length thallium-barium-calcium-copper oxide (TBCCO) or barium-calcium-copper oxide (BCCO) precursor tapes or wires conveyed through the upper chamber to thereby effectuate the deposition of vaporized thallium (being so vaporized as the first reactant in the lower chamber at a temperature between about 700 C and 800 C) on TBCCO or BCCO tape or wire (the second reactant) at its simultaneous annealing temperature in the upper chamber of about 800 to 950 C to thereby replace thallium oxide lost from TBCCO tape or wire because of the high annealing temperature or to deposit thallium on BCCO tape or wire. Continuously moving the tape or wire provides a single-step process that effectuates production of long-length TBCCO superconducting product. 2 figs.

  17. Two chamber reaction furnace

    DOEpatents

    Blaugher, Richard D.

    1998-05-05

    A vertical two chamber reaction furnace. The furnace comprises a lower chamber having an independently operable first heating means for heating the lower chamber and a gas inlet means for admitting a gas to create an ambient atmosphere, and an upper chamber disposed above the lower chamber and having an independently operable second heating means for heating the upper chamber. Disposed between the lower chamber and the upper chamber is a vapor permeable diffusion partition. The upper chamber has a conveyor means for conveying a reactant there through. Of particular importance is the thallinating of long-length thallium-barium-calcium-copper oxide (TBCCO) or barium-calcium-copper oxide (BCCO) precursor tapes or wires conveyed through the upper chamber to thereby effectuate the deposition of vaporized thallium (being so vaporized as the first reactant in the lower chamber at a temperature between about 700.degree. and 800.degree. C.) on TBCCO or BCCO tape or wire (the second reactant) at its simultaneous annealing temperature in the upper chamber of about 800.degree. to 950.degree. C. to thereby replace thallium oxide lost from TBCCO tape or wire because of the high annealing temperature or to deposit thallium on BCCO tape or wire. Continuously moving the tape or wire provides a single-step process that effectuates production of long-length TBCCO superconducting product.

  18. Reactions at Oxygen Atoms

    NASA Astrophysics Data System (ADS)

    Gómez, Ana M.

    Synthetic protocols based on carbohydrates require the differentiation of their abundant hydroxyl groups, by and large, in order to expose just one single hydroxyl group to the selected reagent. This differentiation is usually carried out with the assistance of protecting groups that block the rest of the hydroxyl groups while being compatible with the given reaction conditions. By corollary, the knowledge and apt choice of the appropriate protecting groups is a key factor in successful synthetic endeavors. In this chapter, an overview of the most commonly employed protecting groups in carbohydrate chemistry is given. Alkyl ethers, being robust protecting groups, have a long history in synthetic carbohydrate chemistry and in related structural studies of polysaccharides. Acetals and ketals, which are of fundamental importance in carbohydrate chemistry, are then discussed. Acyl and silyl protecting groups, which also play an important role in modern monosaccharide transformations, are also presented. Finally, recent blocking strategies are described, including orthogonal strategies, by which the protecting groups are harmoniously combined in modern carbohydrate chemistry.

  19. Formaldehyde reactions in dark clouds

    NASA Technical Reports Server (NTRS)

    Sen, A. D.; Anicich, V. G.; Federman, S. R.

    1992-01-01

    The low-pressure reactions of formaldehyde (H2CO) with D(+), D2(+), D3(+), and He(+) are studied by the ion-cyclotron resonance technique. These reactions are potential loss processes for formaldehyde in cores of dark interstellar clouds. The deuterated reactants represent direct analogs for protons. Rate coefficients and branching ratios of product channels have been measured. Charge transfer is observed to be the dominant reaction of H2CO with D(+), D2(+), and He(+) ions. Only the D3(+) reaction exhibits a proton-transfer channel. All reactions proceed at rate coefficients near the collision limit. Proton-deuteron exchange reactions are found to be inefficient processes in the formaldehyde system.

  20. Characterising Complex Enzyme Reaction Data.

    PubMed

    Dönertaş, Handan Melike; Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

  1. Characterising Complex Enzyme Reaction Data

    PubMed Central

    Rahman, Syed Asad; Thornton, Janet M.

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

  2. Speeding chemical reactions by focusing

    NASA Astrophysics Data System (ADS)

    Lacasta, A. M.; Ramírez-Piscina, L.; Sancho, J. M.; Lindenberg, K.

    2013-04-01

    We present numerical results for a chemical reaction of colloidal particles which are transported by a laminar fluid and are focused by periodic obstacles in such a way that the two components are well mixed and consequently the chemical reaction is speeded up. The roles of the various system parameters (diffusion coefficients, reaction rate, and obstacles sizes) are studied. We show that focusing speeds up the reaction from the diffusion limited rate ˜t-1/2 to very close to the perfect mixing rate, ˜t-1.

  3. Anaphylactoid reactions to radiocontrast media.

    PubMed

    Cochran, Sachiko T

    2005-01-01

    Adverse reactions to contrast material are a concern because iodinated contrast materials are commonly used drugs. The risk for adverse reaction is 4% to 12% with ionic contrast materials and 1% to 3% with nonionic contrast materials. The risk for severe adverse reaction is 0.16% with ionic contrast materials and 0.03% with nonionic contrast materials. The death rate, one to three per 100,000 contrast administrations, is similar for both ionic and nonionic agents. More than 90% of adverse reactions with nonionic contrast materials are anaphylactoid. The types of severe reactions seen with nonionic contrast administration were initially predominantly anaphylactoid. With the advent of helical CT angiography, the reactions are now predominantly attributable to cardiopulmonary decompensation. With the widespread use of nonionic contrast materials, adverse reactions are now seen less frequently. Skills involved in evaluating and treating adverse reactions are not as frequently used. Periodic reviews and updates of specific treatment plans for various reactions with the physicians and staff who use contrast material are very important to ensure optimal preparedness. The key to successful treatment is preparation and early intervention. PMID:15659260

  4. Dearomatization Reactions Using Phthaloyl Peroxide.

    PubMed

    Eliasen, Anders M; Christy, Mitchell; Claussen, Karin R; Besandre, Ronald; Thedford, Randal P; Siegel, Dionicio

    2015-09-18

    A new oxidative dearomatization reaction has been developed using phthaloyl peroxide to chemoselectively install two oxygen-carbon bonds into aromatic precursors. The oxidation reaction proceeds only once; addition of superstoichiometric equivalents of phthaloyl peroxide does not react further with the newly generated 1,3-cyclohexadiene. The reaction has been challenged by the addition of different functional groups and shown to maintain chemoselectivity. Due to the broad reactivity with 1,2-methylenedioxybenzene derivatives, linear free energy correlations were determined and support a mechanism proceeding through diradicals analogous to arene-hydroxylation reactions using phthaloyl peroxide. PMID:26333308

  5. (Reaction mechanism studies of heavy ion induced nuclear reactions)

    SciTech Connect

    Mignerey, A.C.

    1991-01-01

    This report discusses the following research projects; decay of excited nuclei formed in La-induced reactions at E/A = 45 MeV; mass and charge distributions in Cl-induced heavy ion reactions; and mass and charge distributions in {sup 56}Fe + {sup 165}Ho at E/A = 12 MeV.

  6. Dynamic Reaction Figures: An Integrative Vehicle for Understanding Chemical Reactions

    ERIC Educational Resources Information Center

    Schultz, Emeric

    2008-01-01

    A highly flexible learning tool, referred to as a dynamic reaction figure, is described. Application of these figures can (i) yield the correct chemical equation by simply following a set of menu driven directions; (ii) present the underlying "mechanism" in chemical reactions; and (iii) help to solve quantitative problems in a number of different…

  7. Entropy Effects in Chelation Reactions.

    ERIC Educational Resources Information Center

    Chung, Chung-Sun

    1984-01-01

    The entropy change for a reaction in aqueous solution can be evaluated as a combination of entropy factors. Valuable insight or understanding can be obtained from a detailed examination of these factors. Several entropy effects of inorganic chemical reactions are discussed as examples. (Author/JN)

  8. Free Radical Reactions in Food.

    ERIC Educational Resources Information Center

    Taub, Irwin A.

    1984-01-01

    Discusses reactions of free radicals that determine the chemistry of many fresh, processed, and stored foods. Focuses on reactions involving ascorbic acid, myoglobin, and palmitate radicals as representative radicals derived from a vitamin, metallo-protein, and saturated lipid. Basic concepts related to free radical structure, formation, and…

  9. The Variance Reaction Time Model

    ERIC Educational Resources Information Center

    Sikstrom, Sverker

    2004-01-01

    The variance reaction time model (VRTM) is proposed to account for various recognition data on reaction time, the mirror effect, receiver-operating-characteristic (ROC) curves, etc. The model is based on simple and plausible assumptions within a neural network: VRTM is a two layer neural network where one layer represents items and one layer…

  10. Chemistry of heavy ion reactions

    SciTech Connect

    Hoffman, D.C.

    1988-10-01

    The use of heavy ions to induce nuclear reactions was reported as early as 1950. Since that time it has been one of the most active areas of nuclear research. Intense beams of ions as heavy as uranium with energies high enough to overcome the Coulomb barriers of even the heaviest elements are available. The wide variety of possible reactions gives rise to a multitude of products which have been studied by many ingenious chemical and physical techniques. Chemical techniques have been of special value for the separation and unequivocal identification of low yield species from the plethora of other nuclides present. Heavy ion reactions have been essential for the production of the trans-Md elements and a host of new isotopes. The systematics of compound nucleus reactions, transfer reactions, and deeply inelastic reactions have been elucidated using chemical techniques. A review of the variety of chemical procedures and techniques which have been developed for the study of heavy ion reactions and their products is given. Determination of the chemical properties of the trans-Md elements, which are very short-lived and can only be produced an ''atom-at-a-time'' via heavy ion reactions, is discussed. 53 refs., 19 figs.

  11. Adverse Reactions to Hallucinogenic Drugs.

    ERIC Educational Resources Information Center

    Meyer, Roger E. , Ed.

    This reports a conference of psychologists, psychiatrists, geneticists and others concerned with the biological and psychological effects of lysergic acid diethylamide and other hallucinogenic drugs. Clinical data are presented on adverse drug reactions. The difficulty of determining the causes of adverse reactions is discussed, as are different…

  12. Enantioselective oxidative boron Heck reactions.

    PubMed

    Lee, A-L

    2016-06-28

    This review highlights the use of the oxidative boron Heck reaction in enantioselective Heck-type couplings. The enantioselective oxidative boron Heck reaction overcomes several limitations of the traditional Pd(0)-catalysed Heck coupling and has subsequently allowed for intermolecular couplings of challenging systems such as cyclic enones, acyclic alkenes, and even site selectively on remote alkenes. PMID:26529247

  13. "Greening up" the Suzuki Reaction

    ERIC Educational Resources Information Center

    Aktoudianakis, Evangelos; Chan, Elton; Edward, Amanda R.; Jarosz, Isabel; Lee, Vicki; Mui, Leo; Thatipamala, Sonya S.; Dicks, Andrew P.

    2008-01-01

    This article describes the rapid, green synthesis of a biaryl compound (4-phenylphenol) via a Pd(0)-catalyzed Suzuki cross-coupling reaction in water. Mild reaction conditions and operational simplicity makes this experiment especially amenable to both mid- and upper-level undergraduates. The methodology exposes students to purely aqueous…

  14. Isosinglet approximation for nonelastic reactions

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.

    1972-01-01

    Group theoretic relations are derived between different combinations of projectile and secondary particles which appear to have a broad range of application in spacecraft shielding or radiation damage studies. These relations are used to reduce the experimental effort required to obtain nuclear reaction data for transport calculations. Implications for theoretical modeling are also noted, especially for heavy-heavy reactions.

  15. Statistical Factors in Complexation Reactions.

    ERIC Educational Resources Information Center

    Chung, Chung-Sun

    1985-01-01

    Four cases which illustrate statistical factors in complexation reactions (where two of the reactants are monodentate ligands) are presented. Included are tables showing statistical factors for the reactions of: (1) square-planar complexes; (2) tetrahedral complexes; and (3) octahedral complexes. (JN)

  16. Tuff reaction vessel experiment

    SciTech Connect

    Bazan, F.; Rego, J.H.

    1986-06-01

    A laboratory leaching test has been performed as part of a project to evaluate the suitability of tuff rocks at Yucca Mountain, Nevada, as a site for a high-level nuclear waste repository. Glass samples of the kind that will be used to store nuclear waste were placed in water inside tuff vessels, and then the tuff vessels were placed in water inside Teflon containers. Glass-component leach rates and migration through the tuff were measured for samples of the ATM-8 actinide glass, which is a PNL 76-68 based glass with low levels of {sup 99}Tc, {sup 237}Np, {sup 238}U, and {sup 239}Pu to simulate wastes. Disc samples of this glass were leached at 90{sup 0}C to 30, 90, and 1983 days inside tuff vessels using a natural groundwater (J-13 well-water) as the leachant. Some samples were held by 304L stainless steel supports to evaluate the effect of this metal on the release rate of glass constituents. At the end of each leaching interval, the J-13 water present inside and outside the rock vessel was analyzed for glass components in solution. On the basis of these analyses, B, Mo, and Tc, appear to migrate through the rock at rates that depend on the porosity of each vessel and the time of reaction. U, Np, and Pu were found only in the inner leachate. Na, Si, and Sr are present in the rock as well as in the J-13 water, and the addition of these elements from the glass could not be determined. Normalized elemental mass loss values for B, Mo, and Tc were calculated using the combined concentrations of the inner and outer leachates and assuming a negligible retention on the rock. The maximum normalized release was 2.3 g/m{sup 2} for Tc. B, Mo, Tc, and Np were released linearly with respect to each other, with B and Mo released at about 85% of the Tc rate, and Np at 5-10% of the Tc rate. Plutonium was found at low levels in the inner leachate but was strongly sorbed on the steel and Teflon supports. Neptunium was sorbed to a lesser extent.

  17. Enzymatic reactions in confined environments

    NASA Astrophysics Data System (ADS)

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

  18. [Anaphylactic reaction following hair bleaching].

    PubMed

    Babilas, P; Landthaler, M; Szeimies, R-M

    2005-12-01

    Ammonium persulphate is a potent bleach and oxidizing agent that is commonly present in hair bleaches. Because bleaching is so commonly performed, hairdressers often develop allergic contact dermatitis to ammonium persulphate. In addition to this delayed reaction, asthma and rhinitis may develop as immediate reactions in those exposed to the fumes. Severe anaphylactic reactions are rare. We report a 24-year-old woman who acquired dermatitis following contact with bleaching substances while working as a hairdresser. After changing her profession, the dermatitis disappeared. Following the private use of a hairdressing bleach containing ammonium persulphate, she suffered a severe anaphylactic reaction with unconsciousness. The patient also developed an anaphylactic reaction three hours following patch testing with the hairdresser battery. The rub test with ammonium persulphate (2.5%) in a 1:100 solution was positive. PMID:15688222

  19. [Skin reactions to tattoo ink].

    PubMed

    Piérard-Franchimont, C; Hermanns, J-F; Piérard, G E

    2011-01-01

    Ritual and artistic tattoos rely on the use of numerous pigments which are not all entirely inert once placed in the dermis. The compositions of some tattoo inks are identified. However, new but less well identified compounds appear on the market. Allergic reactions can be present under different aspects. They may correspond to allergic contact dermatitis or to photodermatitis. Other reactions include allergic hypersensitivity reactions as well as lichenoid, granulomatous or pseudolymphoma reactions. Pulsed light and laser are typically used for regular tattoo removal. These procedures are not indicated in inflamed tattoos. Indeed, the pigment dispersed during photolysis may perpetuate the reaction. Pseudotattoos due to the stratum corneum staining are frequently responsible for photoeczema. PMID:21942077

  20. Fundamental reaction pathways during coprocessing

    SciTech Connect

    Stock, L.M.; Gatsis, J.G.

    1992-12-01

    The objective of this research was to investigate the fundamental reaction pathways in coal petroleum residuum coprocessing. Once the reaction pathways are defined, further efforts can be directed at improving those aspects of the chemistry of coprocessing that are responsible for the desired results such as high oil yields, low dihydrogen consumption, and mild reaction conditions. We decided to carry out this investigation by looking at four basic aspects of coprocessing: (1) the effect of fossil fuel materials on promoting reactions essential to coprocessing such as hydrogen atom transfer, carbon-carbon bond scission, and hydrodemethylation; (2) the effect of varied mild conditions on the coprocessing reactions; (3) determination of dihydrogen uptake and utilization under severe conditions as a function of the coal or petroleum residuum employed; and (4) the effect of varied dihydrogen pressure, temperature, and residence time on the uptake and utilization of dihydrogen and on the distribution of the coprocessed products. Accomplishments are described.

  1. Enzymatic reactions in confined environments.

    PubMed

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems. PMID:27146955

  2. Effective reaction rates for diffusion-limited reaction cycles

    NASA Astrophysics Data System (ADS)

    Nałecz-Jawecki, Paweł; Szymańska, Paulina; Kochańczyk, Marek; Miekisz, Jacek; Lipniacki, Tomasz

    2015-12-01

    Biological signals in cells are transmitted with the use of reaction cycles, such as the phosphorylation-dephosphorylation cycle, in which substrate is modified by antagonistic enzymes. An appreciable share of such reactions takes place in crowded environments of two-dimensional structures, such as plasma membrane or intracellular membranes, and is expected to be diffusion-controlled. In this work, starting from the microscopic bimolecular reaction rate constants and using estimates of the mean first-passage time for an enzyme-substrate encounter, we derive diffusion-dependent effective macroscopic reaction rate coefficients (EMRRC) for a generic reaction cycle. Each EMRRC was found to be half of the harmonic average of the microscopic rate constant (phosphorylation c or dephosphorylation d), and the effective (crowding-dependent) motility divided by a slowly decreasing logarithmic function of the sum of the enzyme concentrations. This implies that when c and d differ, the two EMRRCs scale differently with the motility, rendering the steady-state fraction of phosphorylated substrate molecules diffusion-dependent. Analytical predictions are verified using kinetic Monte Carlo simulations on the two-dimensional triangular lattice at the single-molecule resolution. It is demonstrated that the proposed formulas estimate the steady-state concentrations and effective reaction rates for different sets of microscopic reaction rates and concentrations of reactants, including a non-trivial example where with increasing diffusivity the fraction of phosphorylated substrate molecules changes from 10% to 90%.

  3. Allergic reactions, "spillover' reactions, and T-cell subsets.

    PubMed

    Bruynzeel, D P; Nieboer, C; Boorsma, D M; Scheper, R J; van Ketel, W G

    1983-01-01

    A strong positive, allergic patch-test reaction was elicited in 15 patients with an established allergy for a particular allergen. Patches with a marginally irritating concentration of sodium lauryl sulfate (SLS) were applied at fixed distances. The SLS patch situated adjacent to the allergic reaction was significantly enhanced in 12 of 15 patients (P less than 0.01) compared to more distant SLS reactions ("spillover'). Only quantitative differences were observed in the histologic pictures of the different types of reaction. The infiltrate consisted of lymphocytes and histiocytes, mainly located perivascular in the upper dermis. T-cell subsets were assessed with monoclonal antibodies using an immunoperoxidase technique. The distribution of the different T cells was the same for both reaction types. T cells located outside the perivascular infiltrates (e.g., in the epidermal vesicles) were OKT-8-positive (cytotoxic/suppressor T lymphocytes). Immunofluorescence examination did not show different patterns for the allergic or "enhanced toxic' reactions with regard to the presence of immunoglobulins and complement. The "spillover' phenomenon may cause false-positive patch-test reactions. PMID:6223603

  4. Integrated Microreactors for Reaction Automation: New Approaches to Reaction Development

    NASA Astrophysics Data System (ADS)

    McMullen, Jonathan P.; Jensen, Klavs F.

    2010-07-01

    Applications of microsystems (microreactors) in continuous-flow chemistry have expanded rapidly over the past two decades, with numerous reports of higher conversions and yields compared to conventional batch benchtop equipment. Synthesis applications are enhanced by chemical information gained from integrating microreactor components with sensors, actuators, and automated fluid handling. Moreover, miniaturized systems allow experiments on well-defined samples at conditions not easily accessed by conventional means, such as reactions at high pressure and temperatures. The wealth of synthesis information that could potentially be acquired through use of microreactors integrated with physical sensors and analytical chemistry techniques for online reaction monitoring has not yet been well explored. The increased efficiency resulting from use of continuous-flow microreactor platforms to automate reaction screening and optimization encourages a shift from current batchwise chemical reaction development to this new approach. We review advances in this new area and provide application examples of online monitoring and automation.

  5. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  6. Reactions and properties of clusters

    NASA Astrophysics Data System (ADS)

    Castleman, A. W., Jr.

    1992-09-01

    The elucidation from a molecular point of view of the differences and similarities in the properties and reactivity of matter in the gaseous compared to the condensed state is a subject of considerable current interest. One of the promising approaches to this problem is to utilize mass spectrometry in conjunction with laser spectroscopy and fast-flow reaction devices to investigate the changing properties, structure and reactivity of clusters as a function of the degree of solvation under well-controlled conditions. In this regard, an investigation of molecular cluster ions has provided considerable new insight into the basic mechanisms of ion reactions within a cluster, and this paper reviews some of the recent advances in cluster production, the origin of magic numbers and relationship to cluster ion stabilities, and solvation effects on reactions. There have been some notable advances in the production of large cluster ions under thermal reaction conditions, enabling a systematic study of the influence of solvation on reactions to be carried out. These and other new studies of magic numbers have traced their origin to the thermochemical stability of cluster ions. There are several classes of reaction where solvation has a notable influence on reactivity. A particularly interesting example comes from recent studies of the reactions of the hydroxyl anion with CO2 and SO2, studied as a function of the degree of hydration of OH-. Both reactions are highly exothermic, yet the differences in reactivity are dramatic. In the case of SO2, the reaction occurs at near the collision rate. By contrast, CO2 reactivity plummets dramatically for clusters having more than four water molecules. The slow rate is in accord with observations in the liquid phase.

  7. Nuclear Structure and Reaction Mechanism Studies with Multinucleon Reactions

    SciTech Connect

    Regan, P. H.; Jones, G. A.; Podolyak, Zs.; Abdullah, M.; Gelletly, W.; Langdown, S. D.; Wollel, G.; De Angelis, G.; Gadea, A.; Kroell, Th.; Marginean, N.; Martinez, T.; Napoli, D. R.; Rusu, C.; Tonev, D.; Zhang, Y. H.; Ur, C. A.; Axiotis, M.; Bazzacco, D.; Farnea, E.

    2006-08-14

    This contribution reports on the results of an experiment to study the near-yrast states in selenium- and osmium-like nuclei, following their population in thick-target, multinucleon transfer reactions between an 82Se beam and a 192Os target. The experimental results for the level scheme for 84Se are presented together with investigations into the use of multi-dimensional gamma-ray energy gating to investigate angular momentum population in such heavy-ion binary reactions.

  8. Tilting mode in nuclear reactions

    SciTech Connect

    Dossing, T.; Randrup, J.

    1986-02-01

    The relation between tilting relaxation and the reaction plane dynamics is discussed, providing an intuitive understanding of the expression for the cross section close to the beam direction, which has recently been derived. Second, the tilting relaxation time and the related wriggling relaxation time are discussed, based upon nucleon exchange transport (window friction). Finally, recent experimental information on the tilting mode relaxation is discussed, and the dynamics of the tilting mode is discussed qualitatively for the three different types of nuclear reactions considered, compound nucleus fission, quasifission, and damped nuclear reactions. 9 refs., 3 figs.

  9. Renormalized reaction and relaxation rates

    NASA Astrophysics Data System (ADS)

    Gorbachev, Yuriy E.

    2016-06-01

    Impact of the non-equilibrium on the reaction and relaxation rates (called as generalized relaxation rates - GRR), for the spatially inhomogeneous gas mixture is considered. Discarding the assumption that the 'chemical' part of the collisional integral is a small correction to non-reactive part, the expression for the zero-order GRR is derived. They are represented as a renormalization of the traditional reaction and relaxation rates, which means mixing of all corresponding processes. Thus all reactions and relaxation processes are entangled.

  10. Leukemoid Reaction in Chikungunya Fever

    PubMed Central

    Charaniya, Riyaz; Sahoo, Ratnakar; Tansir, Ghazal; Sasmal, Gargi

    2016-01-01

    Chikungunya is a viral illness caused by an arbovirus which is transmitted by Aedes mosquito. Fever and polyarthralgia are hallmark of this viral illness. Viral infections are generally associated with leucopenia and bacterial infections with leukocytosis. Leukemoid Reaction (LR) is defined by reactive increase in leukocyte count of more than 50,000/cu mm with increase in mature leukocytes on peripheral blood. Leukocytosis is common in Chikungunya but leukemoid reaction has not been reported in medical literature. Our patient presented with high grade fever and symmetrical polyarthritis. Blood investigation showed Leukemoid reaction and after extensive work up a diagnosis of chikungunya was made. PMID:27437276

  11. Catalytic Organometallic Reactions of Ammonia

    PubMed Central

    Klinkenberg, Jessica L.

    2012-01-01

    Until recently, ammonia had rarely succumbed to catalytic transformations with homogeneous catalysts, and the development of such reactions that are selective for the formation of single products under mild conditions has encountered numerous challenges. However, recently developed catalysts have allowed several classes of reactions to create products with nitrogen-containing functional groups from ammonia. These reactions include hydroaminomethylation, reductive amination, alkylation, allylic substitution, hydroamination, and cross-coupling. This Minireview describes examples of these processes and the factors that control catalyst activity and selectivity. PMID:20857466

  12. Hypersensitivity reaction associated with phenytoin

    PubMed Central

    Indu, T. H.; Basutkar, Roopa Satyanarayan

    2015-01-01

    Hypersensitivity reactions with antiepileptic drugs (AEDs) are generally associated with aromatic AEDs. We present a case of hypersensitivity reactions followed by administration of phenytoin with diazepam and ranitidine in a patient with generalized tonic-clonic seizures. Antigen-antibody reactions or decreased levels of epoxide hydrolase are well known with phenytoin. Increased level of serum phenytoin causing toxicities due to competitive inhibition with diazepam on co-administration was also reported in the literature. Prevention of the adverse effects with AEDs is a multi-stage process, which requires implementation of preventive measures through careful monitoring and prompts interventions. PMID:26692739

  13. Magnetically suspended reaction wheel assembly

    NASA Technical Reports Server (NTRS)

    Stocking, G.

    1984-01-01

    The magnetically suspended reaction wheel assembly (MSRWA) is the product of a development effort funded by the Air Force Materials Laboratory (AFML) at Wright Patterson AFB. The specific objective of the project was to establish the manufacturing processes for samarium cobalt magnets and demonstrate their use in a space application. The development was successful on both counts. The application portion of the program, which involves the magnetically suspended reaction wheel assembly, is emphasized. The requirements for the reaction wheel were based on the bias wheel requirements of the DSP satellite. The tasks included the design, fabrication, and test of the unit to the DSP program qualification requirements.

  14. Hypersensitivity reaction associated with phenytoin.

    PubMed

    Indu, T H; Basutkar, Roopa Satyanarayan

    2015-09-01

    Hypersensitivity reactions with antiepileptic drugs (AEDs) are generally associated with aromatic AEDs. We present a case of hypersensitivity reactions followed by administration of phenytoin with diazepam and ranitidine in a patient with generalized tonic-clonic seizures. Antigen-antibody reactions or decreased levels of epoxide hydrolase are well known with phenytoin. Increased level of serum phenytoin causing toxicities due to competitive inhibition with diazepam on co-administration was also reported in the literature. Prevention of the adverse effects with AEDs is a multi-stage process, which requires implementation of preventive measures through careful monitoring and prompts interventions. PMID:26692739

  15. Nuclear reactions at intermediate energies

    NASA Astrophysics Data System (ADS)

    Shyam, Radhey

    2016-05-01

    In the domain of Nuclear reactions at intermediate energies, the QCD coupling constant αs is large enough (~ 0.3 - 0.5) to render the perturbative calculational techniques inapplicable. In this regime the quarks are confined into colorless hadrons and it is expected that effective field theories of hadron interactions via exchange of hadrons, provide useful tools to describe such reactions. In this contribution we discuss the application of one such theory, the effective Lagrangian model, in describing the hadronic reactions at intermediate energies whose measurements are the focus of a vast international experimental program.

  16. Siloxy alkynes in annulation reactions.

    PubMed

    Qian, Hui; Zhao, Wanxiang; Sun, Jianwei

    2014-12-01

    Siloxy alkynes are a family of versatile species in organic synthesis. This account reviews the annulation reactions of siloxy alkynes for the synthesis of a variety of carbo- and heterocyclic products. With various dipolarophiles or dipolarophile-like reaction partners, siloxy alkynes are capable of forming small (three- to six-membered) rings. Recently, we have expanded the scope to the synthesis of medium- and large-ring lactones, enabled by the design of new amphoteric molecules as well as a new ring-expansion strategy. These annulation reactions provide not only practically useful syntheses of cyclic molecules, but also important understanding of the fundamental reactivity of siloxy alkynes. PMID:25171137

  17. Chemical potential and reaction electronic flux in symmetry controlled reactions.

    PubMed

    Vogt-Geisse, Stefan; Toro-Labbé, Alejandro

    2016-07-15

    In symmetry controlled reactions, orbital degeneracies among orbitals of different symmetries can occur along a reaction coordinate. In such case Koopmans' theorem and the finite difference approximation provide a chemical potential profile with nondifferentiable points. This results in an ill-defined reaction electronic flux (REF) profile, since it is defined as the derivative of the chemical potential with respect to the reaction coordinate. To overcome this deficiency, we propose a new way for the calculation of the chemical potential based on a many orbital approach, suitable for reactions in which symmetry is preserved. This new approach gives rise to a new descriptor: symmetry adapted chemical potential (SA-CP), which is the chemical potential corresponding to a given irreducible representation of a symmetry group. A corresponding symmetry adapted reaction electronic flux (SA-REF) is also obtained. Using this approach smooth chemical potential profiles and well defined REFs are achieved. An application of SA-CP and SA-REF is presented by studying the Cs enol-keto tautomerization of thioformic acid. Two SA-REFs are obtained, JA'(ξ) and JA'' (ξ). It is found that the tautomerization proceeds via an in-plane delocalized 3-center 4-electron O-H-S hypervalent bond which is predicted to exist only in the transition state (TS) region. © 2016 Wiley Periodicals, Inc. PMID:27237470

  18. Radiative capture reactions in astrophysics

    SciTech Connect

    Brune, Carl R.; Davids, Barry

    2015-08-07

    Here, the radiative capture reactions of greatest importance in nuclear astrophysics are identified and placed in their stellar contexts. Recent experimental efforts to estimate their thermally averaged rates are surveyed.

  19. Experimental Study of Serpentinization Reactions

    NASA Technical Reports Server (NTRS)

    Cohen, B. A.; Brearley, A. J.; Ganguly, J.; Liermann, H.-P.; Keil, K.

    2004-01-01

    Current carbonaceous chondrite parent-body thermal models [1-3] produce scenarios that are inconsistent with constraints on aqueous alteration conditions based on meteorite mineralogical evidence, such as phase stability relationships within the meteorite matrix minerals [4] and isotope equilibration arguments [5, 6]. This discrepancy arises principally because of the thermal runaway effect produced by silicate hydration reactions (here loosely called serpentinization, as the principal products are serpentine minerals), which are so exothermic as to produce more than enough heat to melt more ice and provide a self-sustaining chain reaction. One possible way to dissipate the heat of reaction is to use a very small parent body [e.g., 2] or possibly a rubble pile model. Another possibility is to release this heat more slowly, which depends on the alteration reaction path and kinetics.

  20. Method for conducting exothermic reactions

    DOEpatents

    Smith, L. Jr.; Hearn, D.; Jones, E.M. Jr.

    1993-01-05

    A liquid phase process for oligomerization of C[sub 4] and C[sub 5] isoolefins or the etherification thereof with C[sub 1] to C[sub 6] alcohols wherein the reactants are contacted in a reactor with a fixed bed acid cation exchange resin catalyst at an LHSV of 5 to 20, pressure of 0 to 400 psig and temperature of 120 to 300 F. wherein the improvement is the operation of the reactor at a pressure to maintain the reaction mixture at its boiling point whereby at least a portion but less than all of the reaction mixture is vaporized. By operating at the boiling point and allowing a portion of the reaction mixture to vaporize, the exothermic heat of reaction is dissipated by the formation of more boil up and the temperature in the reactor is controlled.