Spontaneous Symmetry Breaking and the Renormalization of the Chern-Simons Term

We calculate the one-loop perturbative correction to the coefficient of the \cs term in non-abelian gauge theory in the presence of Higgs fields, with a variety of symmetry-breaking structures. In the case of a residual $U(1)$ symmetry, radiative corrections do not change the coefficient of the \cs term. In the case of an unbroken non-abelian subgroup, the coefficient of the relevant \cs term (suitably normalized) attains an integral correction, as required for consistency of the quantum theory. Interestingly, this coefficient arises purely from the unbroken non-abelian sector in question; the orthogonal sector makes no contribution. This implies that the coefficient of the \cs term is a discontinuous function over the phase diagram of the theory.Comment: Version to be published in Phys Lett B., minor additional change

Methyltransferases from Ruta graveolens L.: Molecular Biology and Biochemistry

The common rue, Ruta graveolens L., is an aromatic plant. It contains acridone alkaloids, furoquinolines, coumarins, and numerous volatile compounds with antimicrobial and allelopathic activity. The starting point of this work was the biosynthesis of acridone alkaloids in Ruta graveolens L., that are restricted to the family of Rutaceae. The N-methylation of anthranilate by the anthranilate N-methyltransferase represents the first step in acridone alkaloid biosynthesis by which the anthranilic acid is removed from the primary metabolism. The anthranilate N-methyltransferase coding cDNA had to be isolated from R. graveolens cell culture material and the recombinant enzyme expressed and characterized. An appropriated method to clone an unknown protein is to isolate it from native tissue followed by microsequencing of the resulted peptides. Specific primers designed from the determined peptides would then be used for the cDNA amplification. In this work, 500 g wet weight of Ruta graveolens R-20 cells grown in suspension culture, were employed for a six-step purification strategy leading to a 450 times purification fold. The gel electrophoresis showed a polypeptide band with an apparent molecular weight of 42-43 kDa. Radioactive mesurements with [Methyl-14C]-SAM showed a high specific activity. The microsequencing of the protein, performed by Dr. Peter Hunziker (Biochemistry Department, University of Zürich), resulted in only five short peptide fragments with no homology with other known methyltransferases. Degenerated oligonucleotide primers, designed based on the peptide fragments, produced a PCR amplicon with no homology to methyltransferases. As the purification did not bring the desired result, degenerated oligonucleotide primers were designed based on the conserved motives (SAM binding site) in O-methyltransferases (OMT). With this method, two full length cDNAs were isolated, R-23 and R-27. R-23 was coding for a 366 amino acid protein with a calculated molecular weight of 40 kDa. R-27 was coding for a 41.6 kDa protein of 374 amino acid residues. These genes were cloned in pQE-60 vector and expressed in E. coli M-15. The protein R-23 had 79% similarity with caffeic acid O-methyltransferases and preserved all the amino acids important for methylation. R-27 showed all the important methyltransferases conserved motifs and had 50% similarity with a putative methyltransferase from Prunus dulcis respectively orcinol and chavicol O-methyltrasnferases from Rosa hybrida. Both bacterial overexpressed proteins were tested for substrate specificity. R-23 coded enzyme did not methylate any of the tested substrates, including caffeic acid. R-27 did methylate 3,5-dimethoxyphenol and other methoxylated phenols. The protein R-27 was purified in four chromatographic steps and characterized. The enzyme showed narrow substrate specificity. The highest affinity was with 3,5-dimethoxyphenol (Km 20.1 µM) with an optimum pH of 7.5. Other accepted substrates were: 3-methoxyphenol, guaiacol, 3,4-dimethoxyphenol and 3,5-dihydroxyanisole. The reaction was independent on cations. The optimum temperature was around 36˚C. On Coomassie stained SDS gel, the purified protein showed a characteristic band of 42 kDa. The calculated molecular weight from a calibrated size exclusion chromatographie was 84 kDa, suggesting that the native enzyme is a homodimer. The reaction product of 3,5-dimethoxyphenol methylation, 1,3,5-trimethoxybenzene is an important component of the scent of roses. Therefore, is reasonable to postulate that the product of the methylation described in this work is a volatile component of the scent of R. graveolens. Based on its substrate specificity, the protein was designated as 3,5-dimethoxyphenol O-methyltransferase. It could be shown that in the presence of Zn2+ the protein efficiently methylate DTT. The product was identified by LC-MS as monomethylthioether. Therefore, 3,5-dimethoxyphenol O-methyltransferase, in addition to the OMT activity, had a thiolmethyltransferase (TMT) activity. Kinetic analysis of 3,5-dimethoxyphenol methylation in the presence of various Zn2+/DTT concentrations showed a competitive DTT binding with an affinity of Ki = 52.0 µM. These results indicate that the OMT and TMT take place in the same active site of the enzyme. Altogether, these results suggest that the substrate specificity of plant O-methyltransferases II and their function in vivo is probably much wider as assumed

Klonierung von O-Methyltransferasen zur Furanocumarinbiosynthese in Ammi majus L.

Vertreter der Apiaceae oder Rutaceae akkumulieren methoxylierte Psoralene wie Bergapten oder Xanthotoxin als Endprodukte der Furanocumarinbiosynthese. O-Methyltransferase-Aktivitäten mit Umsetzungen von Bergaptol zu Bergapten bzw. Xanthotoxol zu Xanthotoxin wurden bereits aus induzierten Zellkultursystemen von Ruta graveolens, Petroselinum crispum und Ammi majus beschrieben. Im Rahmen dieser Arbeit wurde die Bergaptol 5-O-Methyltransferase (BMT) als erstes Enzym der Furanocumarinbiosynthese aus Ammi majus L. in ihrer cDNA und Aminosäuresequenz aufgeklärt und biochemisch charakterisiert. Das translatierte Polypeptid zeigte hohe Sequenzhomologien zu heterologen Kaffeesäure 3-O-Methyltransferasen (COMTs). Daher wurde zum Sequenzvergleich eine COMT aus Ammi majus L. Pflanzen kloniert, die 64% Identität zur BMT auf Aminosäureebene aufweist. Die funktionelle Expression beider Enzyme in Escherichia coli zeigte, dass die BMT Aktivität in bakteriellen Rohextrakten labil ist und beim Anreinigen verloren geht. Die COMT Aktivität dahingegen bleibt stabil. Während die BMT eine hohe Spezifität für ihr Substrat Bergaptol aufweist, methyliert die COMT 5-Hydroxyferulasäure, Esculetin und weitere Substrate. Die BMT-Sequenz aus Ammi majus stellt einen neuen spezifischen Zugang auf molekularer Ebene zur Biosynthese der Furanocumarine dar

Transformation of acridone synthase to chalcone synthase

AbstractAcridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal reactions in the formation of acridone alkaloids or flavonoids. While acridone alkaloids are confined almost exclusively to the Rutaceae, flavonoids occur abundantly in all seed-bearing plants. ACSs and CHSs had been cloned from Ruta graveolens and shown to be closely related polyketide synthases which use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA, respectively, as the starter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2-pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunological cross reactivities and molecular modeling studies suggested an almost identical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity towards CHS activity, and site-directed mutagenesis replacing all three residues by the corresponding amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe) fully transformed the enzyme to a functional CHS with comparatively marginal ACS activity. The results suggested that ACS divergently has evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity has developed in the Rutaceae only

Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase

Abstract Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3b-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1 H NMR and CD spectroscopies as (À)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional nonspecific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.

Исследование парогазовой установки, работающей на генераторном газе

Выпускная квалификационная работа 116 страниц, 25 рисунков, 25 таблиц, 35 источников, 2 приложения. Ключевые слова: газификация угля, парогазовая установка, газотурбинная установка, генераторный газ, анализ, котел-утилизатор, показатели эффективности. Объектом исследования является ПГУ, работающая на генераторном газе. Цель работы – анализ работоспособности такой ПГУ с ГТУ, работающей на генераторном газе. В процессе исследования проводилось изучение различных способов газификации угля; производился выбор расчетной схемы ПГУ с газификацией; проводился расчет состава и характеристик генераторного газа и определение основных показателей тепловой эффективности ПГУ. В результате исследования был сделан анализ эффективности работы парогазовой установки.Final qualifying work of 116 pages, 25 figures, 25 tables, 35 sources, 2 annexes. Keywords: coal gasification combined cycle plant, a gas turbine plant, the product gas analysis, waste heat boiler, performance indicators. The object of this study is to CCGT running on syngas. The purpose of work - performance analysis of a CCGT with a gas turbine, operating on syngas. The study was carried out to study different methods of coal gasification; selects the estimated PSU circuit with gasification; conducted calculation and composition of the product gas characteristics and the definition of the main indicators of the thermal efficiency of the CCGT. The survey was made the analysis of the efficiency of the combined-cycle plant

Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (؉)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9 -10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (؉)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (؉)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (؉)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (؉)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution. Furanocoumarins are produced by many plants, mostly of the Apiaceae, Rutaceae, Moraceae, or the Coronilla and Psoralea genera of the Fabaceae (1-3). Multiple pharmacological effects have been ascribed to several of these metabolites (4 -6), which were included in clinical screenings but received attention also for their inhibitory effect on monooxygenases involved in drug metabolism (7-9) and potential toxicity (10). The (dihydro)furan-substituted 2H-1-benzopyran-2-one forms the characteristic core structure, and the annulation type distinguishes the linear furanocoumarins or psoralens from the angular furanocoumarin

Significant Short-Term Shifts in the Microbiomes of Smokers With Periodontitis After Periodontal Therapy With Amoxicillin & Metronidazole as Revealed by 16S rDNA Amplicon Next Generation Sequencing

The aim of this follow-up study was, to compare the effects of mechanical periodontal therapy with or without adjunctive amoxicillin and metronidazole on the subgingival microbiome of smokers with periodontitis using 16S rDNA amplicon next generation sequencing. Fifty-four periodontitis patients that smoke received either non-surgical periodontal therapy with adjunctive amoxicillin and metronidazole (n = 27) or with placebos (n = 27). Subgingival plaque samples were taken before and two months after therapy. Bacterial genomic DNA was isolated and the V4 hypervariable region of the bacterial 16S rRNA genes was amplified. Up to 96 libraries were normalized and pooled for Illumina MiSeq paired-end sequencing with almost fully overlapping 250 base pairs reads. Exact ribosomal sequence variants (RSVs) were inferred with DADA2. Microbial diversity and changes on the genus and RSV level were analyzed with non-parametric tests and a negative binomial regression model, respectively. Before therapy, the demographic, clinical, and microbial parameters were not significantly different between the placebo and antibiotic groups. Two months after the therapy, clinical parameters improved and there was a significantly increased dissimilarity of microbiomes between the two groups. In the antibiotic group, there was a significant reduction of genera classified as Porphyromonas, Tannerella, and Treponema, and 22 other genera also decreased significantly, while Selenomonas, Capnocytophaga, Actinomycetes, and five other genera significantly increased. In the placebo group, however, there was not a significant decrease in periodontal pathogens after therapy and only five other genera decreased, while Veillonella and nine other genera increased. We conclude that in periodontitis patients who smoke, microbial shifts occurred two months after periodontal therapy with either antibiotics or placebo, but genera including periodontal pathogens decreased significantly only with adjunctive antibiotics

Организация работ по охране труда и оценка рисков работников нефтегазодобывающего предприятия

Несмотря на усовершенствование технологических процессов и вводимые меры по обеспечению безопасности труда, на предприятиях существует тенденция к снижению себестоимости продукции. В связи с этим увеличивается вероятность возникновения производственных рисков на промышленных предприятиях, которые рассмотрены в данной работе на примере нефтегазодобывающего управления "Федоровскнефть".Despite the improvement of technological processes and the introduction of measures to ensure labor safety, there is a tendency at enterprises to reduce the cost of production. In this connection, the probability of occurrence of industrial risks in industrial enterprises is increased, which are considered in this work by the example of oil and gas production department "Fedorovskneft"