- Studies on the reaction between reduced riboflavin and selenocystine
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Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys59-S-S-Cys64, and finally (iii) from Cys59 and Cys64 to the selenosulfide bond Cys497-S-Se-Sec498. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec-Sec), an oxidized form of Sec. The interaction between RibH2 and Sec-Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec-Sec was (7.9?±?0.1)?×?10?2?M?1 s?1 (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec-Rib complex, followed by nucleophilic attack of Sec-Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2?±?0.1)?×?106?M?1 (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond.
- Dereven'kov, Ilia A.,Makarov, Sergei V.,Molodtsov, Pavel A.,Makarova, Anna S.
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p. 146 - 153
(2020/09/21)
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- A high-throughput screening for inhibitors of riboflavin synthase identifies novel antimicrobial compounds to treat brucellosis
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Brucella spp. are pathogenic intracellular Gram-negative bacteria adapted to life within cells of several mammals, including humans. These bacteria are the causative agent of brucellosis, one of the zoonotic infections with the highest incidence in the world and for which a human vaccine is still unavailable. Current therapeutic treatments against brucellosis are based on the combination of two or more antibiotics for prolonged periods, which may lead to antibiotic resistance in the population. Riboflavin (vitamin B2) is biosynthesized by microorganisms and plants but mammals, including humans, must obtain it from dietary sources. Owing to the absence of the riboflavin biosynthetic enzymes in animals, this pathway is nowadays regarded as a rich resource of targets for the development of new antimicrobial agents. In this work, we describe a high-throughput screening approach to identify inhibitors of the enzymatic activity of riboflavin synthase, the last enzyme in this pathway. We also provide evidence for their subsequent validation as potential drug candidates in an in?vitro brucellosis infection model. From an initial set of 44?000 highly diverse low molecular weight compounds with drug-like properties, we were able to identify ten molecules with 50% inhibitory concentrations in the low micromolar range. Further Brucella culture and intramacrophagic replication experiments showed that the most effective bactericidal compounds share a 2-Phenylamidazo[2,1-b][1,3]benzothiazole chemical scaffold. Altogether, these findings set up the basis for the subsequent lead optimization process and represent a promising advancement in the pursuit of novel and effective antimicrobial compounds against brucellosis.
- Serer, María Inés,Carrica, Mariela del Carmen,Trappe, J?rg,López Romero, Sandra,Bonomi, Hernán Ruy,Klinke, Sebastián,Cerutti, María Laura,Goldbaum, Fernando Alberto
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p. 2522 - 2535
(2019/04/17)
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- Site-Selective Synthesis of 15N- and 13C-Enriched Flavin Mononucleotide Coenzyme Isotopologues
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Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.
- Neti, Syam Sundar,Poulter, C. Dale
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p. 5087 - 5092
(2016/07/06)
-
- Single label comparative hybridization
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The present invention provides methods of detecting and mapping chromosomal or genetic abnormalities associated with various diseases or with predisposition to various diseases, or to detecting the phenomena of large scale copy number variants. In particular, the present invention provides advanced methods of performing array-based comparative hybridization that allow reproducibility between samples and enhanced sensitivity by using the same detectable label for both test sample and reference sample nucleic acids. Invention methods are useful for the detection or diagnosis of particular disease conditions such as cancer, and detecting predisposition to cancer based on detection of chromosomal or genetic abnormalities and gene expression level. Invention methods are also useful for the detection or diagnosis of hereditary genetic disorders or predisposition thereto, especially in prenatal samples. Moreover, invention methods are also useful for the detection or diagnosis of de novo genetic aberrations associated with post-natal developmental abnormalities.
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- A solid-state pH sensor for nonaqueous media including ionic liquids
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We describe a solid state electrode structure based on a biologically derived proton-active redox center, riboflavin (RFN). The redox reaction of RFN is a pH-dependent process that requires no water. The electrode was fabricated using our previously described 'stuffing' method to entrap RFN into vapor phase polymerized poly(3,4-ethylenedioxythiophene). The electrode is shown to be capable of measuring the proton activity in the form of an effective pH over a range of different water contents including nonaqueous systems and ionic liquids (ILs). This demonstrates that the entrapment of the redox center facilitates direct electron communication with the polymer. This work provides a miniaturizable system to determine pH (effective) in nonaqueous systems as well as in ionic liquids. The ability to measure pH (effective) is an important step toward the ability to customize ILs with suitable pH (effective) for catalytic reactions and biotechnology applications such as protein preservation.
- Thompson, Brianna C.,Winther-Jensen, Orawan,Winther-Jensen, Bjorn,Macfarlane, Douglas R.
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p. 3521 - 3525
(2013/05/22)
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- Nucleic acid size detection method
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The present invention provides methods of determining the size of a particular nucleic acid segment of interest in a sample of nucleic acids through fragmentation of DNA, size fractionation, an optional second fragmentation, and identification using a marker sequence. In particular aspects, an expansion or reduction of tandem repeat sequences can be detected. In further aspects, carriers and individuals afflicted with fragile X syndrome or other diseases associated with tandem repeats can be distinguished from normal individuals.
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- O-nucleoside, S-nucleoside, and N-nucleoside probes of lumazine synthase and riboflavin synthase
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Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction.
- Talukdar, Arindam,Zhao, Yujie,Lv, Wei,Bacher, Adelbert,Illarionov, Boris,Fischer, Markus,Cushman, Mark
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experimental part
p. 6239 - 6261
(2012/09/25)
-
- Aerobic reduction of olefins by in situ generation of diimide with synthetic flavin catalysts
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A versatile reducing agent, diimide, can be generated efficiently by the aerobic oxidation of hydrazine with neutral and cationic synthetic flavin catalysts 1 and 2. This technique provides a convenient and safe method for the aerobic reduction of olefins, which proceeds with 1 equiv of hydrazine under an atmosphere of O2 or air. The synthetic advantage over the conventional gas-based method has been illustrated through high hydrazine efficiency, easy and safe handling, and characteristic chemoselectivity. Vitamin B2 derivative 6 acts as a highly practical, robust catalyst for this purpose because of its high availability and recyclability. Association complexes of 1b with dendritic 2,5-bis(acylamino)pyridine 15 exhibit unprecedented catalytic activities, with the reduction of aromatic and hydroxy olefins proceeding significantly faster when a higher-generation dendrimer is used as a host pair for the association catalysts. Contrasting retardation is observed upon similar treatment of non-aromatic or non-hydroxy olefins with the dendrimer catalysts. Control experiments and kinetic studies revealed that these catalytic reactions include two independent, anaerobic and aerobic, processes for the generation of diimide from hydrazine. Positive and negative dendrimer effects on the catalytic reactions have been ascribed to the specific inclusion of hydrazine and olefinic substrates into the enzyme-like reaction cavities of the association complex catalysts. Copyright
- Imada, Yasushi,Iida, Hiroki,Kitagawa, Takahiro,Naota, Takeshi
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body text
p. 5908 - 5920
(2011/07/07)
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- Oxygen reactivity in flavoenzymes: Context matters
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Many flavoenzymes-oxidases and monooxygenases-react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 105 M-1 s-1 for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M-1 s-1, suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O2 with rate constants of 6.2 × 104 and 3.0 × 103 M-1 s-1, respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as those for the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O2.
- McDonald, Claudia A.,Fagan, Rebecca L.,Collard, Francois,Monnier, Vincent M.,Palfey, Bruce A.
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supporting information; experimental part
p. 16809 - 16811
(2011/12/04)
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- Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase
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Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a Ki of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π-π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.
- Talukdar, Arindam,Morgunova, Ekaterina,Duan, Jianxin,Meining, Winfried,Foloppe, Nicolas,Nilsson, Lennart,Bacher, Adelbert,Illarionov, Boris,Fischer, Markus,Ladenstein, Rudolf,Cushman, Mark
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experimental part
p. 3518 - 3534
(2010/08/05)
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- Facile oxidation of leucomethylene blue and dihydroflavins by artemisinins: Relationship with flavoenzyme function and antimalarial mechanism of action
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The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH2 initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH2 for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA-MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of twoelectron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH2 in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure-activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox-active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon-centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron-carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra-erythrocytic human disulfide reductases required for maintenance of parasite redox balance.
- Haynes, Richard K.,Chan, Wing-Chi,Wong, Ho-Ning,Li, Ka-Yan,Wu, Wai-Keung,Fan, Kit-Man,Sung, Herman H. Y.,Williams, Ian D.,Prosperi, Davide,Melato, Sergio,Coghi, Paolo,Monti, Diego
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experimental part
p. 1282 - 1299
(2011/01/04)
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- Substractive single label comparative hybridization
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Provided are methods of determining differences between nucleic acids in a test sample and a reference sample. In certain embodiments the methods are used for detecting and mapping chromosomal or genetic abnormalities associated with various diseases or with predisposition to various diseases, or to detecting the phenomena of large scale copy number variants. In particular, provided are advanced methods of performing array-based comparative hybridization that allow reproducibility between samples and enhanced sensitivity by using the same detectable label for both test sample and reference sample nucleic acids. Invention methods are useful for the detection or diagnosis of particular disease conditions such as cancer, and detecting predisposition to cancer based on detection of chromosomal or genetic abnormalities and gene expression level. Invention methods are also useful for the detection or diagnosis of hereditary genetic disorders or predisposition thereto, especially in prenatal samples. Moreover, invention methods are also useful for the detection or diagnosis of de novo genetic aberrations associated with post-natal developmental abnormalities.
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- A new series of N-[2,4-dioxo-6-D-ribitylamino-1,2,3,4-tetrahydropyrimidin- 5-yl]oxalamic acid derivatives as inhibitors of lumazine synthase and riboflavin synthase: Design, synthesis, biochemical evaluation, crystallography, and mechanistic implications
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(Figure Presented) The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
- Zhang, Yanlei,Illarionov, Boris,Morgunova, Ekaterina,Jin, Guangyi,Bacher, Adelbert,Fischer, Markus,Ladenstein, Rudolf,Cushman, Mark
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p. 2715 - 2724
(2008/09/19)
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- Light Emitting Probes
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This invention relates to a composition comprising at least two chemically different fluorophores, providing a donor and an acceptor respectively, connected together by at least one linker moiety and bonded to a binder moiety.
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- A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D- ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: Design, synthesis, and evaluation
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(Chemical Equation Presented) Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly.
- Zhang, Yanlei,Jin, Guangyi,Illarionov, Boris,Bacher, Adelbert,Fischer, Markus,Cushman, Mark
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p. 7176 - 7184
(2008/02/12)
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- Multiconstituent liquid lgG and IgM calibrators
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A serum in which are dissolved at least two heterologous antibodies, which are independently IgG or IgM and which specifically bind to different antigens, serves as a calibrator for multi-analyte immunoassays.
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- Oligonucleotides comprising a molecular switch
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This invention relates to oligonucleotides comprising a molecular switch which may exist in an “open” or “closed” position. The molecular switch portion of the probe is particularly sensitive to the identity of sequences complementary to the molecular switch. Oligonucleotides containing a molecular switch are applicable to all kinds of hybridization processes. Due to the sensitivity of the switch domain of the oligonucleotide, probes containing a molecular switch are particularly useful in the identification of single point mismatches. More specifically, a portion, but not all, of the oligonucleotide becomes unbound from a mismatched target. The invention further relates to methods of using said oligonucleotides for research reagents, and clinical diagnostics. An exemplary oligonucleotide comprises a first hybridizable domain, a second bridging block domain, and a third binding domain.
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- Nutritional supplement
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A nutritional supplement is provided that is designed to provide nutritional benefits as well as to assist the body with detoxification. By providing a supplement that serves both of these functions, the present invention may enable persons to improve their overall wellness.
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- Dual resonance energy transfer nucleic acid probes
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Dual nucleic acid probes with resonance energy transfer moieties are provided. In particular, fluorescent or luminescent resonance energy transfer moieties are provided on hairpin stem-loop molecular beacon probes that hybridize sufficiently near each other on a subject nucleic acid, e.g. mRNA, to generate an observable interaction. The invention also provides lanthanide chelate luminescent resonance energy transfer moieties on linear and stem-loop probes that hybridize sufficiently near each other on a subject nucleic acid to generate an observable interaction. The invention thereby provides detectable signals for rapid, specific and sensitive hybridization determination in vivo. The probes are used in methods of detection of nucleic acid target hybridization for the identification and quantification of tissue and cell-specific gene expression levels, including response to external stimuli, such as drug candidates, and genetic variations associated with disease, such as cancer.
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- Incorporation of an amide into 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase
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Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase. The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction. The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase. Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors. Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings. However, molecular modeling of one of the inhibitors with E. coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.
- Cushman, Mark,Yang, Donglai,Mihalic, Jeffrey T.,Chen, Jinhua,Gerhardt, Stefan,Huber, Robert,Fischer, Markus,Kis, Klaus,Bacher, Adelbert
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p. 6871 - 6877
(2007/10/03)
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- Compositions and methods for prevention and treatment of chronic diseases and disorders including the complications of diabetes mellitus
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This invention relates to nutrient and therapeutic compositions for treatment and prevention of symptoms and disease conditions associated with microangiopathy and macroangiopathy and to methods using the compositions. In particular, the invention relates to compositions useful in the treatment of diabetic retinopathy and nephropathy, to compositions useful in the treatment of other retinal disorders including macular degeneration and cataracts, to compositions useful in wound healing, to compositions useful for treatment and prevention of neuropathy, to compositions useful for treatment and prevention of cardiovascular disease and to compositions useful for the treatment and prevention of dental and periodontal disorders.
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- DNA encoding human κ casein and process for obtaining the protein
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The present invention relates to an expression system comprising a DNA sequence encoding a polypeptide which has a biological activity of human κ-casein, the system comprising a 5′-flanking sequence capable of mediating expression of said DNA sequence. In preferred embodiments the 5′-flanking sequence is from a milk protein gene of a mammal such as a casein gene or whey acidic protein (WAP) gene and the DNA sequence contains at least one intron sequence selected from the intron sequences presented in SEQ ID NO:30. The invention also relates to DNA sequences, replicable expression vectors and cells harboring said vectors, recombinant polypeptide e.g. in glycosylated form, and milk, infant formula or nutrient supplement comprising recombinant polypeptide. The invention further relates to a transgenic non-human mammal such as a mouse, rat, rabbit, goat, sheep, pig, lama, camel or bovine species whose germ cells and somatic cells contain a DNA sequence as defined above as a result of chromosomal incorporation into the non-human mammalian genome, or into the genome of an ancestor of said non-human mammal.
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- Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon
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The present invention provides labeled nucleic acid amplification oligonucleotides, which can be linear or hairpin primers or blocking oligonucleotides. The oligonucleotides of the invention are labeled with donor and/or acceptor moieties of molecular energy transfer pairs. The moieties can be fluorophores, such that fluorescent energy emitted by the donor is absorbed by the acceptor. The acceptor may be a fluorophore that fluoresces at a wavelength different from the donor moiety, or it may be a quencher. The oligonucleotides of the invention are configured so that a donor moiety and an acceptor moiety are incorporated into the amplification product. The invention also provides methods and kits for directly detecting amplification products employing the nucleic acid amplification primers. When labeled linear primers are used, treatment with exonuclease or by using specific temperature eliminates the need for separation of unincorporated primers. This "closed-tube" format greatly reduces the possibility of carryover contamination with amplification products, provides for high throughput of samples, and may be totally automated.
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- Mechanistic implications of a linear free-energy correlation of rate constants for the reduction of active- and Met-R2 forms of E. coli ribonucleotide reductase with eight organic radicals
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Cross-reaction rate constants k12 (22 °C) at pH 7.0 have been determined for the reduction of Fe(III)2 and tyrosyl-radical-containing active-R2 from E. coli ribonucleotide reductase with eight organic radicals (OR), e.g., MV·+ from methyl viologen. The more reactive OR's were generated in situ using pulse radiolysis (PR) techniques, and other OR's were generated by prior reduction of the parent with dithionite, followed by stopped-flow (SF) studies. In both procedures it was necessary to include consideration of doubly-reduced parent forms. Values of k12 are in the range 109 to 104 M-1 s-1 and reduction potentials E1/(o) for the OR vary from -0.446 to +0.l94 V. Samples of E. coli active-R2 also have an Fe2(III) met-R2 component (with no Tyr·), which in the present work was close to 40%. From separate experiments met-R2 gave similar k12 rate constants (on average 66% bigger) to those for active-R2, suggesting that reduction of the Fe2/(III) center is the common rate-limiting step. A single Marcus free-energy plot of log k12 - 0.5 log f vs - E1/(o)/0.059 describes all the data, and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. It is concluded that the ratelimiting step involves electron transfer. In addition, the intercept at -E1/(o)/0.059 = 0 is 5.94, where values of the reduction potential and self-exchange rate constant for met-R2 contribute to this value. To maintain electroneutrality at the ~10 A buried active site H+ uptake is also required. For both e- and H+ transfer the conserved pathway Trp-48, Asp-237, His-118 to Fe(A) is a possible candidate requiring further examination.
- Dobbing, A. Mark,Borman, Christopher D.,Twitchett, Mark B.,Leese, David N.,Salmon, G. Arthur,Sykes, A. Geoffrey
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p. 2206 - 2212
(2007/10/03)
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- Reduction of Hg(II)·EDTA by conformationally biased flavins
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The rates of Hg(II)·EDTA reduction by conformationally biased flavins are reported. The rates of the reduction correlate to the redox potential of the flavin model. In the case of 8,9,10-trimethylflavin (6), the rate was slower than expected from the redox potential.
- Hasford, Justin J.,Rizzo, Carmelo J.
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p. 1317 - 1320
(2007/10/03)
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- Gastric preparation with sustained release
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A gastric preparation developed in order to solve the technical problems of conventional preparations, which is prepared by the bilayer packing technique and comprises 5 to 60%, desirably 10 to 40% of a rapid release portion which can establish the therapeutic level of a drug shortly after the administration and 95 to 40%, desirably 90 to 60% of a sustained release portion which has a specific gravity or 1 or less and can maintain a satisfactory release rate.
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- Chemistry of peroxidic tetrahedral intermediates of flavin
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By means of pulse radiolysis 4a-peroxy intermediates of normal and 5-alkylated flavins were produced and the kinetics of their decay into flavin and the corresponding hydroperoxide was investigated as a function of the pH. The neutral and proton-catalyzed breakdown of the 4a-intermediates of 5-alkylated flavinium cations on the one hand and of 5-protio flavins on the other was very similar. It was concluded that the rate-determining step in the neutral decomposition of normal flavin 4a-peroxides is a heterolysis along the C(4a)-O bond which is catalyzed by water as a general acid. The species initially produced consist of a N(5)-protonated flavinium cation, a neutral hydroperoxide, and a hydroxide ion. The process is completed by rapid deprotonation of the flavinium cation to yield the neutral flavin. By combination of kinetic and thermodynamic data determined in this and other laboratories, the energetics of the autoxidation of 1,5-dihydroflavin was resolved into individual steps. The proton-catalyzed breakdown of flavin 4a-peroxides is initiated by a proton-assisted expulsion of neutral hydroperoxide leaving behind the N(5)-protonated flavinium cation. The attenuation of proton catalysis with decreasing pH indicates thermodynamic protonation of the 4a-intermediates around pH 3. The site of protonation is presumably the N(5) or the N(10) atom. The hydroxide ion catalyzed breakdown of the 4a-species is best interpreted by assuming the rate-determining step to be deprotonation of the N(S)-H site followed by rapid expulsion of the hydroperoxide anion and neutral flavin. This picture demands the microscopic pKa of the N(5)-H group to be below 17. The possible role of enzymes in stabilizing the 4a-intermediates against breakdown into flavin and hydroperoxide is discussed. It is suggested that an apolar, hydrophobic pocket may be the chief stabilizing factor. In such an environment, the transition state for heterolysis and homolysis may approach each other. Finally, the bond strength of the peroxidic O-O bond was calculated from recent thermodynamic data. This bond turns out to be weaker (26 kcal/mol) than the O-O bond in any known linear peroxide. From the finding that the O-O bond is weaker than the C(4a)-O bond it is argued that, in sufficiently hydrophobic enzymes, monooxygenation may be initiated by homolysis of the O-O bond. It is suggested that the comparable strengths of the C(4a)-O and O-O bonds may be the prime reason for the versatility of flavin enzymes.
- Merényi, Gábor,Lind, Johan
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p. 3146 - 3153
(2007/10/02)
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- Biosynthesis of Riboflavin. A Simple Synthesis of the Substrate and Product of the Pyrimidine Deaminase and of Structural Analogs
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2-Amino-5-nitro-6-ribitylamino-4(3H)-pyrimidinone (7) was phosphorylated with chlorophosphoric acid yielding an isomer mixture containing about 63percent of the 5'-phosphate 7a together with other monophosphates and biphosphates of 7.Preparative HPLC afforded pure 7a.Catalytic hydrogenation of 7a yields the labile substrate of pyrimidine deaminase, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate (2a).The product of the enzyme, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (4a), can be obtained from 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (8) by an analogous procedure.The 3'- and 4'-monophosphates of 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are not substrates for the deaminase. 5'-Phosphates of a variety of ribityl-substituted pyrimidines and pteridines could also be obtained by phosphorylation with chlorophosphoric acid without the use of protecting groups. - Keywords: Riboflavin, Biosynthesis, Pyrimidine, Deaminase, 5'-Phosphates of Ribitylaminopyrimidines
- Nielsen, P.,Bacher, A.
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p. 1358 - 1364
(2007/10/02)
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- LAMPTEROMYCES BIOLUMINESCENCE - 2 LAMPTEROFLAVIN, A LIGHT EMITTER IN THE LUMINOUS MUSHROOM, L. japonicus
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A new fluorescent compound, lampteroflavin, was isolated from a luminous mushroom (Lampteromyces japonicus).Its properties suggest that it has a function as the light emitter in the mushroom bioluminescence.Its structure was assigned to be pentofuranosyl riboflavin, 1.
- Isobe, Minoru,Uyakul, Duangchan,Goto, Toshio
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p. 1169 - 1172
(2007/10/02)
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- Preparation of riboflavin, and 4,5-dimethyl-N-(D)-ribityl-2-(O-alkoxyphenylazo)-aniline intermediates
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An improved process for the preparation of riboflavin by condensation of a 4,5-dimethyl-N-(D)-ribityl-2-phenylazoaniline derivative with barbituric acid in the presence of an acidic condensing agent in an organic solvent, wherein a 4,5-dimethyl-N-(D)-ribityl-2-(o-alkoxyphenylazo)-aniline of the formula IIa STR1 where R is alkyl of 1 to 4 carbon atoms, in particular methyl, is reacted with barbituric acid, as well as the novel intermediates of the formula IIa.
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- Preparation of riboflavin
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Riboflavin of the formula I STR1 is prepared by condensing a 4,5-dimethyl-N-(D)-ribityl-2-phenylazoaniline of the formula II STR2 where R is H or --Cl, --NO2 or --CH3 in the o- or p-position, with barbituric acid of the formula III STR3 in the presence of an acid as the condensing agent, by an improved process in which the acidic condensing agent used is an aliphatic or cycloaliphatic/aliphatic tertiary carboxylic acid of the general formula IV STR4 where R1, R2 and R3 are each a lower alkyl group, R1, R2 and R3 together containing 3 to 20, preferably 3 to 10, carbon atoms, or R1 is a lower alkyl group, in particular methyl, and R2 and R3 together form a tetramethylene or pentamethylene group. The process can be particularly advantageously carried out using trimethylacetic acid or a commercial mixture of saturated tertiary carboxylic acids, e.g. Versatic R 10-acid.
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- Biosynthesis of Riboflavin. Enzymatic Deamination of 2,5-Diamino-6-hydroxy-4-ribitylaminopyrimidine-5'-phosphate
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Cell extracts of Saccharomyces cerevisiae and Candida guilliermondii catalyze the hydrolytic deamination in position 2 of 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine-5'-phosphate.The deaminase activity is absent in rib 2 mutants of S. cerevisiae.These findings confirm the biosynthetic pathway proposed earlier on the basis of metabolic and genetic studies. - Keywords: Riboflavin Biosynthesis, Deaminase, 2,5-Diamino-6-hydroxy-4-ribitylaminopyrimidine-5'-phosphate
- Klein, Gerhard,Bacher, Adelbert
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p. 482 - 484
(2007/10/02)
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