Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms

Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of ~88 and ~87kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III)OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.     

Regulation of lactic acid production during exercise

Lactic acid accumulates in contracting muscle and blood beginning at approximately 50-70% of the maximal O2 uptake, well before the aerobic capacity is fully utilized. The classical explanation has been that part of the muscle is O2 deficient and therefore lactate production is increased to provide supplementary anaerobically derived energy. Currently, however, the predominant view is that lactate production during submaximal dynamic exercise is not O2 dependent. In the present review, data and arguments in support of and against the hypothesis of O2 dependency have been scrutinized. Data underlying the conclusion that lactate production during exercise is not O2 dependent were found to be 1) questionable, or 2) interpretable in an alternative manner. Experiments in human and animal muscles under various conditions demonstrated that the redox state of the muscle is reduced (i.e., NADH is increased) either before or in parallel with increases in muscle lactate. Based on experimental data and theoretical considerations, it is concluded that lactate production during submaximal exercise is O2 dependent. The amount of energy provided through the anaerobic processes during steady-state submaximal exercise is, however, low, and the role of lactate formation as an energy source is of minor importance. It is proposed that the achievement of increased aerobic energy formation under conditions of limiting O2 availability requires increases of ADP, Pi, and NADH and that the increases in ADP (and therefore AMP via the adenylate kinase equilibrium) and Pi will stimulate glycolysis, and the resulting increase in cytosolic NADH will shift the lactate dehydrogenase equilibrium toward increased lactate production.     

NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Third‐generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long‐read sequences. This is not only advantageous for genome sequencing projects, but also advantageous for amplicon‐based high‐throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high‐quality consensus sequences. Here, we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long‐read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

The conserved active site asparagine in class I ribonucleotide reductase is essential for catalysis.

The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate. To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover but promoted rapid protein R2 tyrosyl radical decay in the presence of the k(cat) inhibitor 2'-azido-2'-deoxy-CDP with similar decay rate constants as the wild-type R1. These results show that all Asn-437 mutants can perform 3'-H abstraction, the first substrate-related step in the reaction mechanism. The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP. The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2'-OH group in the form of water, a step that has been proposed to drive the 3'-H abstraction step. A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step. We conclude that Asn-437 is essential for catalysis but not for 3'-H abstraction. We propose that the suicidal N437A, N437S, and N437D mutants can also catalyze the water elimination step, whereas the inert N437Q mutant cannot. Our results suggest that Asn-437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.     

Cytotoxicity testing of wound-dressing materials

A method was developed for testing the cytotoxicity of various bandage-like wound dressings and gel wound dressings. In this method, the ability of human polymorphonuclear neutrophils (PMNs) to initiate a respiratory burst after exposure to the various wound dressings is used as a marker of cytotoxicity. Luminol-amplified chemiluminescence stimulated with opsonised zymosan or phorbol 12-myristate 13-acetate (PMA) is used to measure the degree of activation of the respiratory burst, i.e. the NADPH oxidase activity, after exposure to wound dressings. Opsonised zymosan (material from yeast cell walls) is a phagocytic stimulus that activates the NADPH oxidase by binding to FC-receptors and complement receptors, and functions as an artificial bacterium, whereas PMA activates the NADPH oxidase by direct activation of protein kinase C. NADPH oxidase activity was inhibited by several wound dressings. The down-regulation of the respiratory burst is detrimental to the bactericial effect of PMNs, and can be used as a marker for the cytotoxicity of wound dressing materials.     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.     

Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition

We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.