Mutational analysis of the hydantoin hydrolysis pathway in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> RU-KM3<Subscript>S</Subscript>

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding d-amino acids or l-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase (N-carbamoylase). Pseudomonas putida strain RU-KM3_S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and l-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and -ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not -ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or -ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3_S cells.     

Theoretical investigation of reactivities of amines in the <Emphasis Type="Italic">N</Emphasis>-nitrosation reactions by N<Subscript>2</Subscript>O<Subscript>3</Subscript>

N-nitrosamine is a class of carcinogenic, mutagenic, and teratogenic compounds, which can be produced from N-nitrosation of amine by nitrosating agents. N-nitrosation of 19 amines (eight acyclic amines, five heterocyclic amines, and six amines with unsaturated groups) by N₂O₃ was investigated at the CBS-QB3 level of theory. The results indicate that generally the heterocyclic amines have the highest reactivities among the three kinds of amines, whereas the reactivities of the amines with unsaturated and electron-withdrawing groups are relatively low. Frontier molecular orbital analysis indicates that the energy gap between the HOMO of an amine and the LUMO of N₂O₃ has a close connection with the reactivity of an amine. A structure-reactivity relationship of amines in the N-nitrosation reactions by N₂O₃ was established using the stepwise multivariate linear regression. The results indicate that the reactivity of an amine has a definite relationship (R_adj² = 0.947) with the heterolytic bond dissociation energy of R₁R₂N-H bond, energy of HOMO, NBO occupancy of the natural lone pair orbital of N atom, the NBO charge of the N atom, and the pyramidalization angle of an amine. This work will be helpful to gain more insight into the N-nitrosation reactions.     

On the Manner of Cyclization of <Emphasis Type="Italic">N</Emphasis>-Acylated Aspartic and Glutamic Acid Derivatives

Abstract  

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and ¹H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives.     

N<Subscript>2</Subscript>O and CH<Subscript>4</Subscript> emission from <Emphasis Type="Italic">Miscanthus</Emphasis> energy crop fields in the infertile Loess Plateau of China

Background

The greenhouse gas (GHG) mitigation is one of the most important environmental benefits of using bioenergy replacing fossil fuels. Nitrous oxide (N₂O) and methane (CH₄) are important GHGs and have drawn extra attention for their roles in global warming. Although there have been many works of soil emissions of N₂O and CH₄ from bioenergy crops in the field scale, GHG emissions in large area of marginal lands are rather sparse and how soil temperature and moisture affect the emission potential remains unknown. Therefore, we sought to estimate the regional GHG emission based on N₂O and CH₄ releases from the energy crop fields.

Results

Here we sampled the top soils from two Miscanthus fields and incubated them using a short-term laboratory microcosm approach under different conditions of typical soil temperatures and moistures. Based on the emission measurements of N₂O and CH₄, we developed a model to estimate annual regional GHG emission of Miscanthus production in the infertile Loess Plateau of China. The results showed that the N₂O emission potential was 0.27 kg N ha^?1 year^?1 and clearly lower than that of croplands and grasslands. The CH₄ uptake potential was 1.06 kg C ha^?1 year^?1 and was slightly higher than that of croplands. Integrated with our previous study on the emission of CO₂, the net greenhouse effect of three major GHGs (N₂O, CH₄ and CO₂) from Miscanthus fields was 4.08 t CO_2eq ha^?1 year^?1 in the Loess Plateau, which was lower than that of croplands, grasslands and shrub lands.

Conclusions

Our study revealed that Miscanthus production may hold a great potential for GHG mitigation in the vast infertile land in the Loess Plateau of China and could contribute to the sustainable energy utilization and have positive environmental impact on the region.

     

Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Nitrate and flooding induce N<Subscript>2</Subscript>O emissions from soybean nodules

Nitrous oxide (N₂O) is one of the three main biogenic greenhouse gases (GHGs) and agriculture represents close to 30 % of the total N₂O net emissions. In agricultural soils, N₂O is emitted by two main microbial processes, nitrification and denitrification, both of which can convert synthetic nitrogen fertilizer into N₂O. Legume-rhizobia symbiosis could be an effective and environmental-friendly alternative to nitrogen fertilization and hence, to mitigate soil N₂O emissions. However, legume crops also contribute to N₂O emissions. A better understanding of the environmental factors involved in the emission of N₂O from nodules would be instrumental for mitigating the release of this GHG gas. In this work, in vivo N₂O emissions from nodulated soybean roots in response to nitrate (0, 1, 2 and 4 mM) and flooding have been measured. To investigate the contribution of rhizobial denitrification in N₂O emission from nodules, plants were inoculated with B. japonicum USDA110 and napA and nosZ denitrification mutants. The results showed that nitrate was essential for N₂O emissions and its concentration enhanced N₂O fluxes showing a statistical linear correlation, being the highest N₂O fluxes obtained with 4 mM nitrate. When inoculated plants grown with 4 mM nitrate were subjected to flooding, a 150- and 830-fold induction of N₂O emission rates from USDA110 and nosZ nodulated roots, respectively, was observed compared to non-flooded plants, especially during long-term flooding. Under these conditions, N₂O emissions from detached nodules produced by the napA mutant were significantly lower (p?<?0.05) than those produced by the wild-type strain (382 versus 1120 nmol N₂O h^?1 g^?1 NFW, respectively). In contrast, nodules from plants inoculated with the nosZ mutant accumulated statistically higher levels of N₂O compared to wild-type nodules (2522 versus nmol 1120 N₂O h^?1 g^?1 NFW, p?<?0.05). These results demonstrate that flooding is an important environmental factor for N₂O emissions from soybean nodules and that B. japonicum denitrification is involved in such emission.     

Rapid degradation of <Emphasis Type="Italic">N</Emphasis>-3-oxo-acylhomoserine lactones by a <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">cereus</Emphasis> isolate from Malaysian rainforest soil

A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 μg AHL h⁻¹ per 10⁹ CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif ₁₀₆HXDH-59 amino acids-H₁₆₉-21 amino acids-D₁₉₁ for N-acylhomoserine lactone lactonases.     

Method for resolution and quantification of components of the non-photochemical quenching (<Emphasis Type="Italic">q</Emphasis><Subscript>N</Subscript>)

A new method of the chlorophyll (Chl) a fluorescence quenching analysis is described, which allows the calculation of values of (at least) three components of the non-photochemical quenching of the variable Chl a fluorescence (q _N) using a non-linear regression of a multi-exponential function within experimental data. Formulae for coefficients of the “energy”-dependent (ΔpH-dependent) quenching (q _E), the state-transition quenching (q _T) and the photo/inhibitory quenching (q _I) of Chl a fluorescence were found on the basis of three assumptions: (i) the dark relaxation kinetics of q _N, as well as of all its components, is of an exponential nature, (ii) the superposition principle is valid for individual Chl a fluorescence quenching processes and (iii) the same reference fluorescence level (namely the maximum variable Chl a fluorescence yield in the dark-adapted state, F _V) is used to define both q _N and its components. All definitions as well as the algorithms for analytical recognition of the q _N components are theoretically clarified and experimentally tested. The described theory results in a rather simple equation allowing to compute values for all q _N components (q _E, q _T, q _I) as well as the half-times of relaxation (τ_1/2) of corresponding quenching processes. It is demonstrated that under the above assumptions it holds: q _N = q _E + q _T + q _I. The theoretically derived equations are tested, and the results obtained are discussed for non-stressed and stressed photosynthetically active samples. Semi-empirical formulae for a fast estimation of values of the q _N components from experimental data are also given.     

Nitrous oxide (N<Subscript>2</Subscript>O) and subsequent open-label SSRI treatment of adolescents with depression (NOTAD): study protocol for a randomised controlled trial

Background

The first line of pharmacological treatment for severe depressive disorders in young people is selective serotonin reuptake inhibitors (SSRIs). However, beneficial clinical effects are rarely observed before several weeks into treatment. Nitrous oxide (N₂O) has a long-standing safety record for pain relief and has been used in adults and young people. In adults with severe treatment-resistant depression, a single dose of N₂O had significant antidepressant effects, with maximum antidepressant effects observed 24 h after administration. However, the antidepressant effects of N₂O have never been investigated in adolescents with a confirmed diagnosis of depression in a prospective trial. The aims of this study are to (1) investigate whether a single inhaled N₂O administration leads to antidepressant effects in adolescents with depression at 24 h, (2) determine whether combined N₂O and SSRI administration (commenced after N₂O intervention) provides a clinically significant improvement in mood over and above the benefits from SSRI administration alone, and, (3) investigate whether the effect seen following N₂O administration can be used as a predictor of SSRI treatment response.

Methods/design

In this study, we will use a single-blind, randomised, placebo-controlled design. Patients aged between 12 and 17 years with major depressive disorder will be recruited. This study will consist of two phases: phase A and phase B. During phase A, participants will be randomised to receive either inhaled N₂O or placebo (air) for 1 h. In phase B, participants will receive open-label pharmacological treatment with the SSRI fluoxetine and will be followed over a 12-week period. Participants will undertake mood assessments at 2 and 24 h after N₂O or placebo administration (phase A) and weekly during the 12-week follow up in phase B.

Discussion

We expect an antidepressant effect from a single dose of inhaled N₂O compared with placebo at 24 h after administration. Additionally, we expect that subjects treated with N₂O will also show greater improvements than the placebo group after 6 and 12 weeks into fluoxetine treatment because of potential additive antidepressant effects. Such findings would be of clinical importance because currently children and adolescents often do not experience any symptom alleviation for several weeks following the initiation of SSRIs.

Trial registration

Australian and New Zealand Clinical Trials Registry, ACTRN12616001568404. Registered on 14 November 2016.

     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

The adsorption of NO,NH<Subscript>3</Subscript>, N<Subscript>2</Subscript> on carbon surface: a density functional theory study

To explore the adsorption mechanism of NO, NH₃, N₂ on a carbon surface, and the effect of basic and acidic functional groups, density functional theory was employed to investigate the interactions between these molecules and carbon surfaces. Molecular electrostatic potential, Mulliken population analyses, reduced density gradient, and Mayer bond order analyses were used to clarify the adsorption mechanism. The results indicate that van der Waals interactions are responsible for N₂ physisorption, and N₂ is the least likely to adsorb on a carbon surface. Modification of carbon materials to decorate basic or acidic functional groups could enhance the NH₃ physisorption because of hydrogen bonding or electrostatic interactions, however, NO physisorption on a carbon surface is poor. Zig-zag sites are more reactive than armchair sites when these gas molecules absorb on the edge sites of carbon surface.

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Graphical abstract NH₃, N₂, NO adsortion on carbon surface

     

Effect of nitrogen forms on growth,cell composition and N<Subscript>2</Subscript> fixation of <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis> in phosphorus-limited chemostat cultures

The aim of this research was to test whether NH₄ ⁺ and NO₃ ⁻ affect the growth, P demand, cell composition and N₂ fixation of Cylindrospermopsis raciborskii under P limitation. Experiments were carried out in P-limited (200 μg l⁻¹ PO₄-P) chemostat cultures of C. raciborskii using an inflowing medium containing either 4,000 μg l⁻¹ NH₄-N, 4,000 μg l⁻¹ NO₃-N or no combined N. The results showed the cellular N:P and C:P ratios of C. raciborskii decreased towards the Redfield ratio with increasing dilution rate (D) due to the alleviation of P limitation. The cellular C:N and carotenoids:chlorophyll-a ratios also decreased with D, predominantly as a result of an increase in the chlorophyll-a and N content. The NH₄ ⁺ and NO₃ ⁻ supply reduced the P maintenance cell quota of C. raciborskii. Consequently, the biomass yield of the N₂-grown culture was significantly lower. The maximum specific growth rate of N₂-grown culture was also the lowest observed. It is suggested that these differences in growth parameters were caused by the P and energy requirement for heterocyte formation, nitrogenase synthesis and N₂ fixation. N₂ fixation was partially inhibited by NO₃ ⁻ and completely inhibited by NH₄ ⁺. It was probably repressed through the high N content of cells at high dissolved N concentrations. These results indicate that C. raciborskii is able to grow faster and maintain a higher biomass under P limitation where a sufficient supply of NH₄ ⁺ or NO₃ ⁻ is maintained. Information gained about the species-specific nutrient and pigment stoichiometry of C. raciborskii could help to access the degree of nutrient limitation in water bodies. Handling editor: Luigi Naselli-Flores     

Facile Synthesis of <Emphasis Type="Italic">N</Emphasis>-protected Amino Acid Esters Assisted by Microwave Irradiation

A highly efficient and safe methodology for synthesis of various N-protected amino acid ethyl esters have been established in this study. This methodology employs orthoesters as both esterification reagent and solvent for protected amino acids. The reactions were carried out under microwave irradiation in neutral conditions for only 2 min, resulting in highly pure crude products in most cases. This strategy works with a variety of N-protecting groups, such as acid labile protecting group: BOC and tBu; base labile protecting group: Fmoc; hydrogenation labile protecting group: Z and Na/NH₃ labile protecting group: Tos, thus providing facile access to numerous valuable building blocks for solid phase synthesis. Further reduction of the crude protected amino acid ethyl ester by sodium borohydride under mild conditions led to the corresponding protected β-amino alcohols with excellent yield, as demonstrated by three examples.     

Chemical Modification of Insulin by <Emphasis Type="Italic">N</Emphasis>-phosphorylation

In this paper, the reactions of bovine insulin and small peptides, such as actin binding domain of thymosin β₄ and Growth Hormone Releasing Factor (GRF 1–29 amino acids) with diisopropyloxyphosphite (DIPPH) and dimethyloxyphosphite (DMPH) were studied by modified Todd reaction. The MALDI-TOF or ESI-MS results showed that lysine, histidine and arginine residues in insulin could be phosphorylated under the water/ethanol system. The N,N,N-diisopropyloxyphosphorylated insulin analogues were characterized using MALDI-TOF and ³¹P NMR. These insulin analogues with different phosphorylation degree were separated and identified through LC-ESI-MS. In addition, circular dichroism (CD) spectra showed that the conformation of N,N,N-dimethyloxyphosphorylated insulin were only changed a little, whereas, that of N,N,N-diisopropyloxyphosphorylated insulin was changed completely.     

N<Subscript>2</Subscript> fixation in three perennial <Emphasis Type="Italic">Trifolium</Emphasis> species in experimental grasslands of varied plant species richness and composition

This study is the first to investigate quantitative effects of plant community composition and diversity on N₂ fixation in legumes. N₂ fixation in three perennial Trifolium species grown in field plots with varied number of neighbouring species was evaluated with the ¹⁵N natural abundance method (two field sites, several growing seasons, no N addition) and the isotope dilution method (one site, one growing season, 5 g N m⁻²). The proportion of plant N derived from N₂ fixation, pNdfa, was generally high, but the N addition decreased pNdfa, especially in species-poor communities. Also following N addition, the presence of grasses in species-rich communities increased pNdfa in T. hybridum and T. repens L., while legume abundance had the opposite effect. In T. repens, competition for light from grasses appeared to limit growth and thereby the amount of N₂ fixed at the plant level, expressed as mg N₂ fixed per sown seed. We conclude that the occurrence of diversity effects seems to be largely context dependent, with soil N availability being a major determinant, and that species composition and functional traits are more important than species richness regarding how neighbouring plant species influence N₂ fixation in legumes.     

Theoretical study of N<Subscript>4</Subscript>X (X = O,S, Se) systems

A series of N₄X (X = O, S, Se) compounds have been examined with ab initio and density functional theory (DFT) methods. To our knowledge, these compounds, except for the C_2v ring and the C_3v towerlike isomers of N₄O, are first reported here. The ring structures are the most energetically favored for N₄X (X = O and S) systems. For N₄Se, the cagelike structure is the most energetically favored. Several decomposition and isomerization pathways for the N₄X species have been investigated. The dissociation of C_2v ring N₄O and N₄S structures via ring breaking and the barrier height are only 1.1 and −0.2 kcal mol⁻¹ at the CCSD(T)/6-311+G*//MP2/6-311+G* level of theory. The dissociation of the cagelike N₄X species is at a cost of 12.1–16.2 kcal mol⁻¹. As for the towerlike and triangle bipyramidal isomers, their decomposition or isomerization barrier heights are all lower than 10.0 kcal mol⁻¹. Although the C_S cagelike N₄S isomer has a moderate isomerization barrier (18.3–29.1 kcal mol⁻¹), the low dissociation barrier (−1.0 kcal mol⁻¹) indicates that it will disappear when going to the higher CCSD(T) level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.