Oxidation of Indole and Indole Derivatives Catalyzed by Nonheme Chloroperoxidases

Indole, indolylacetic acid, and tryptophan were oxidized by chloroperoxidases isolated from strains of Streptomyces lividansand Pseudomonas pyrrocinia. Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were isolated from the reaction medium.     

Purine Derivatives of 1,2-Disubstituted Cyclohexane Analogues of Nucleosides

Abstract

Starting from (±)-cis-2-hydroxymethylcyclohexylamine, a series of cyclohexane-derived cis-1,2-disubstituted carbonucleoside analogues with a 6- or 2,6-purine or 8-azapurine base were synthesized. The antiviral and antitumoral in vitro effects of the new compounds were evaluated.     

Peroxynitrite Mediated Oxidation of Purine Bases of Nucleosides and Isolated DNA

Reaction of nitric oxide with superoxide anion produces the highly reactive species peroxynitrite (ONOO^?). This compound has been shown to be a strong oxidant of lipids and proteins. However, no data are available on its effect on DNA, with the exception of the induction of strand breaks. We report the result of studies on the reactions of peroxynitrite with the adenine and guanine moieties of nucleosides and isolated DNA. The samples were analyzed for 8-oxo-7,8-dihydro-2′-deoxyguano-sine (8-oxo-dGuo), 2,2-diamino-4–[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5–(2H)-oxazolone (oxazolone) and 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-dAdo). The effects of peroxynitrite treatment were compared with those of ionizing radiation in aerated aqueous solution, chosen as a source of hydroxyl radicals. At the nucleoside level, both oxidizing conditions led to the formation of oxazolone and 8-oxo-dAdo. In addition, evidence was provided for the formation of the 4R* and 4S* diastereoisomers of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine. The latter dGuo oxidation products were chosen as markers of the release of singlet oxygen (¹O₂) upon reaction of peroxynitrous acid with hydrogen peroxide. Oxidation of purine bases was then studied within isolated DNA. A significant increase in the level of 8-oxp-dGuo, oxazolone and 8-oxo-dAdo was observed within double stranded DNA upon exposure to γ-radiation. Oxazolone and 8-oxo-dAdo were formed upon peroxynitrite treatment but no significant increase in the amount of 8-oxo-dGuo was detected. These results showed that peroxynitrite exhibits oxidizing properties toward purine moieties both in nucleosides and isolated DNA. However, the significant differences in the oxidative damage distribution within DNA observed after exposure to γ radiation by comparison with peroxynitrite treatment questions the involvement of hydroxyl radicals as the main oxidizing species released by decomposition of peroxynitrous acid.     

Bacteriology of Manganese Nodules: II. Manganese Oxidation by Cell-free Extract from a Manganese Nodule Bacterium

A cell-free extract from Arthrobacter 37, isolated from a manganese nodule from the Atlantic Ocean, exhibited enzymatic activity which accelerated manganese accretion to synthetic Mn-Fe oxide as well as to crushed manganese nodule. The reaction required oxygen and was inhibited by HgCl₂ and p-chloromercuribenzoate but not by Atebrine dihydrochloride. The rate of enzymatic action depended on the concentration of cell-free extract used. The enzymatic activity had a temperature optimum around 17.5 C and was destroyed by heating at 100 C. The amount of heat required for inactivation depended on the amount of nucleic acid in the preparation. In the cell-free extract, unlike the whole-cell preparation, peptone could not substitute for NaHCO₃ in the reaction mixture. An enzyme-containing protein fraction and a nucleic acid fraction could be separated from cell extract by gel filtration, when prepared in 3% NaCl but not in seawater. The nucleic acid fraction was not required for enzymatic activity.     

Synthesis And Biological Evaluation Of Nitrogen Mustard Derivatives Of Purine Bases

This paper deals with the synthesis of nitrogen mustard analogs, derivatives of purine bases. Alkylation in position N-9 and diethanolamine fixation on position 6 were managed by microwave irradiations. Chlorination of these dihydroxylated intermediates led to a cyclization, giving tricyclic purine base analogs bearing a chloroethyl chain. Finally, MTT assays on obtained compounds do not show cytotoxicity on four different cancer cell lines.     

嘌呤核苷及其衍生物的代谢工程

嘌呤核苷及其衍生物被广泛应用于食品和医药领域。利用诱变筛选技术可以获得嘌呤核苷类产品的工业生产菌株,但往往耗时,效率低,而且获得的某些高产菌株还存在不稳定的缺陷。菌株代谢调控与生理生化的研究为代谢工程优化嘌呤核苷类产品的合成提供了理论基础,利用代谢工程改造菌株合成嘌呤核苷也引起了研究人员的关注。系统地介绍了微生物嘌呤生物合成途径及其调控机制,综述了嘌呤核苷类产品及其衍生物的代谢工程研究进展,最后讨论了利用代谢工程改造菌株合成这些产品面临的问题及今后的研究方向。     

Kinetics of Manganese Oxidation by Cell-Free Extracts of Bacteria Isolated from Manganese Concretions from Soil

A study of the kinetics of Mn²⁺ oxidation catalyzed by cell extracts of two bacterial isolates (E₁, Pseudomonas III [new isolate] and E₄, Citrobacter freundii) isolated from the core of manganese concretions from Greek soils is presented. The reaction velocity of Mn²⁺ oxidation was determined from the rate of consumption of Mn²⁺. The oxidation of Mn²⁺ was followed by measuring changes in Mn²⁺ concentration by activation analysis and by atomic absorption spectrophotometry. The reaction velocity was directly proportional to cell extract concentration when the reaction time was 1 h. At longer reaction times, the relationship deviated from linearity because substrate concentration became limiting. The rate of Mn²⁺ oxidation increased with the Mn²⁺ concentration. Analysis of the results by application of the integrated Michaelis equation for determining Michaelis constants and maximal velocities either in the presence (K_m = 3.33 μmol/ml and V_max = 1.25 μmol/ml·h) or in the absence of maleate buffer (K_m = 2.52 μmol/ml and V_max = 2.04 μmol/ml·h) indicated a strong affinity between the oxidizing system and manganese. All results in this study are consistent with an enzymatic manganese-oxidizing system and give an indication of the mechanism of biological Mn²⁺ oxidation in soil which differs from that in the marine environment.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

The Synthesis of Derivatives of 2-Arylamino ara-Carbocyclic Purine Nucleosides as Potential Anti-Viral Agents

Abstract

A general synthetic method into 2-arylamino ara-carbocyclic purine nucleosides from 2,4,6-mchloropyrimidine is described.     

Oxidation of Benzidine and Its Derivatives by Thyroid Peroxidase

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.     

Thirteen Years Experience with Selective Screening for Disorders in Purine and Pyrimidine Metabolism

Purine and pyrimidine disorders represent a heterogeneous group with variable clinical symptoms and low prevalence rate. In the last thirteen years, we have studied urine/plasma specimens from about 1600 patients and we have identified 35 patients: eight patients with adenylosuccinate lyase deficiency, eight patients with hypoxanthine-guanine phosphoribosyltransferase deficiency, one patient with purine nucleoside phosphorylase deficiency, ten patients with xanthine dehydrogenase deficiency, six patients with molybdenum cofactor deficiency and two patients with dihydropyrimidine dehydrogenase deficiency.

Despite low incidence of these diseases, our findings highlight the importance of including the purine and pyrimidine analysis in the selective screening for inborn errors of metabolism in specialized laboratories, where amino acid and organic acid disorders are simultaneously investigated.     

c-Type Cytochromes and Manganese Oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Manganese Oxidation by Bacterial Isolates from the Indian Ridge System

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA = 2 gm l⁻¹ nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl₂·4H₂O were in the order of 10⁶ CFU l⁻¹. Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 × 10⁶ CFU l⁻¹. The retrievable counts on Mn amended dilNA ranged from ND to 3.21× 10⁶, 1.47 × 10⁶ and 1.45 × 10⁶ CFU l⁻¹ on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n = 39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR–Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 × 10⁶ CFU l⁻¹), while CR48 represented only 6% of the total CFU (1.05 × 10⁶ CFU l⁻¹). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 μM Mn both in unamended and amended media. At levels of Mn greater than 100 μM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 μM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 μM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn ammendments from 1 μM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (≥1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 μM Mn day⁻¹ in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 μM Mn day⁻¹ in unamended medium and 35 μM Mn day⁻¹ in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices ≈2 μm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7–10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.     

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract

The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized.

Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction