The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures

Addition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6-h batch experiments, the initial NH3-N uptake rates were physiologically accelerated by a factor of 1.4-13. NH2OH addition caused a 20-40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3-N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order-of-magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH-related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions

Assimilation of atmospheric CO₂ by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.     

厌氧氨氧化菌的代谢途径及其关键酶的研究进展

作为新型生物脱氮工艺的代表,厌氧氨氧化反应的代谢功能菌——厌氧氨氧化(Anammox)菌也逐渐成为研究的热点。本文介绍了10种Anammox菌的发现过程,阐述了Anammox菌的关键酶的研究进展。通过对Candidatus"Brocadia anammoxidans"和Candidatus"Kuenenia stuttgartiensis"菌的代谢途径的分析和推理,综述了化学计量模型、生化反应模型和能量代谢模型,并提出了一种可能存在的基于关键酶的厌氧氨氧化菌微界面代谢新途径。     

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration.

Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(III) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamine but did not provide protection.

The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O^·-₂ and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) Involvement of the PGR5–PGRL1 pathway under anaerobic conditions

In oxygenic photosynthesis, cyclic electron flow around photosystem I denotes the recycling of electrons from stromal electron carriers (reduced nicotinamide adenine dinucleotide phosphate, NADPH, ferredoxin) towards the plastoquinone pool. Whether or not cyclic electron flow operates similarly in Chlamydomonas and plants has been a matter of debate. Here we would like to emphasize that despite the regulatory or metabolic differences that may exist between green algae and plants, the general mechanism of cyclic electron flow seems conserved across species. The most accurate way to describe cyclic electron flow remains to be a redox equilibration model, while the supramolecular reorganization of the thylakoid membrane (state transitions) has little impact on the maximal rate of cyclic electron flow. The maximum capacity of the cyclic pathways is shown to be around 60 electrons transferred per photosystem per second, which is in Chlamydomonas cells treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and placed under anoxic conditions. Part I of this work (aerobic conditions) was published in a previous issue of BBA-Bioenergetics (vol. 1797, pp. 44–51) (Alric et al., 2010).     

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.     

Stability of hydroxylamino- and amino-intermediates from reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene

Hydroxylamines, produced as intermediates in the reductive metabolism of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene between nitroaromatic parent compounds and corresponding amines, were unstable in aqueous solution in the presence of O₂. Reactions of hydroxylamines to compounds other than amines may be the major cause of poor mass balance observations in bioremediation systems where only aminated products are monitored. Results demonstrate the formation of azoxy compounds as products of abiotic aryl-hydroxylamine reactions.     

羟胺切割Neurturin融合蛋白表达载体的构建、表达产物纯化及生物活性

 Neurturin (NTN)是新近发现的一种神经营养因子 ,是 GDNF家族的成员之一 .将 5′端引入了羟胺切割位点的 h NTN基因克隆到硫氧还蛋白融合表达载体 p Thio His A,在宿主菌 BL2 1中获得了稳定、高效表达 ,表达产物以包涵体形式存在 .在变性条件下经羟胺切割、柱层析纯化后复性 ,获得纯度达 90 %以上的 rh NTN.经鸡胚背根神经节 (DRG)培养法测定具有生物学活性 .     

Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA

Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively. Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.