Feeding dicyandiamide (DCD) to cattle: An effective method to reduce N2O emissions from urine patches in a heavy-textured soil under temperate climatic conditions.

Nitrate (NO3-) leaching and nitrous oxide (N2O) emission from urine patches in grazed pastures are key sources of water and air pollution, respectively. Broadcast spraying of the nitrification inhibitor dicyandiamide (DCD) has been shown to reduce these losses, but it is expensive. As an alternative, it had been demonstrated that feeding DCD to cattle (after manual mixing with supplementary feeds) was a practical, effective and cheaper method to deliver high DCD rates within urine patches. This two-year study carried out on simulated urine patches in three application seasons (spring, summer, autumn) explored the efficacy of DCD feeding to cattle to reduce N losses from grazed pasture soil in a heavy-textured soil under temperate climatic conditions. In each application season, DCD fed to cows, then excreted with urine and applied at a rate of 30kgDCDha-1 (treatment U+DCD30-f) was as effective as powdered DCD mixed with normal urine and applied at the same rate (treatment U+DCD30) at reducing cumulative N2O-N emissions and the N2O-N emission factor (EF3, expressed as % of N applied). Increasing DCD loading within urine patches from 10 to 30kgDCDha-1 improved efficacy by significantly reducing the EF3 from 34% to 64%, which highlights that under local conditions, 10kgDCDha-1 (the recommended rate for commercial use in New Zealand) was not the optimum DCD rate to curb N2O emissions. The modelling of EF3 in this study also suggests that N mitigation should be given more attention when soil moisture is going to be high, which can be predicted with short-term weather forecasting. DCD feeding, for instance in autumn when cows are not lactating and the risk of N losses is high, could then be reduced by focusing mainly on those forecasted wet periods.

Combining stable isotopes with contamination indicators: A method for improved investigation of nitrate sources and dynamics in aquifers with mixed nitrogen inputs.

Excessive nitrate (NO3-) concentration in groundwater raises health and environmental issues that must be addressed by all European Union (EU) member states under the Nitrates Directive and the Water Framework Directive. The identification of NO3- sources is critical to efficiently control or reverse NO3- contamination that affects many aquifers. In that respect, the use of stable isotope ratios 15N/14N and 18O/16O in NO3- (expressed as δ15N-NO3- and δ18O-NO3-, respectively) has long shown its value. However, limitations exist in complex environments where multiple nitrogen (N) sources coexist. This two-year study explores a method for improved NO3- source investigation in a shallow unconfined aquifer with mixed N inputs and a long established NO3- problem. In this tillage-dominated area of free-draining soil and subsoil, suspected NO3- sources were diffuse applications of artificial fertiliser and organic point sources (septic tanks and farmyards). Bearing in mind that artificial diffuse sources were ubiquitous, groundwater samples were first classified according to a combination of two indicators relevant of point source contamination: presence/absence of organic point sources (i.e. septic tank and/or farmyard) near sampling wells and exceedance/non-exceedance of a contamination threshold value for sodium (Na+) in groundwater. This classification identified three contamination groups: agricultural diffuse source but no point source (D+P-), agricultural diffuse and point source (D+P+) and agricultural diffuse but point source occurrence ambiguous (D+P±). Thereafter δ15N-NO3- and δ18O-NO3- data were superimposed on the classification. As δ15N-NO3- was plotted against δ18O-NO3-, comparisons were made between the different contamination groups. Overall, both δ variables were significantly and positively correlated (p < 0.0001, rs = 0.599, slope of 0.5), which was indicative of denitrification. An inspection of the contamination groups revealed that denitrification did not occur in the absence of point source contamination (group D+P-). In fact, strong significant denitrification lines occurred only in the D+P+ and D+P± groups (p < 0.0001, rs > 0.6, 0.53 ≤ slope ≤ 0.76), i.e. where point source contamination was characterised or suspected. These lines originated from the 2-6‰ range for δ15N-NO3-, which suggests that i) NO3- contamination was dominated by an agricultural diffuse N source (most likely the large organic matter pool that has incorporated 15N-depleted nitrogen from artificial fertiliser in agricultural soils and whose nitrification is stimulated by ploughing and fertilisation) rather than point sources and ii) denitrification was possibly favoured by high dissolved organic content (DOC) from point sources. Combining contamination indicators and a large stable isotope dataset collected over a large study area could therefore improve our understanding of the NO3- contamination processes in groundwater for better land use management. We hypothesise that in future research, additional contamination indicators (e.g. pharmaceutical molecules) could also be combined to disentangle NO3- contamination from animal and human wastes.

Improving and disaggregating N2O emission factors for ruminant excreta on temperate pasture soils.

Cattle excreta deposited on grazed grasslands are a major source of the greenhouse gas (GHG) nitrous oxide (N2O). Currently, many countries use the IPCC default emission factor (EF) of 2% to estimate excreta-derived N2O emissions. However, emissions can vary greatly depending on the type of excreta (dung or urine), soil type and timing of application. Therefore three experiments were conducted to quantify excreta-derived N2O emissions and their associated EFs, and to assess the effect of soil type, season of application and type of excreta on the magnitude of losses. Cattle dung, urine and artificial urine treatments were applied in spring, summer and autumn to three temperate grassland sites with varying soil and weather conditions. Nitrous oxide emissions were measured from the three experiments over 12months to generate annual N2O emission factors. The EFs from urine treated soil was greater (0.30-4.81% for real urine and 0.13-3.82% for synthetic urine) when compared with dung (-0.02-1.48%) treatments. Nitrous oxide emissions were driven by environmental conditions and could be predicted by rainfall and temperature before, and soil moisture deficit after application; highlighting the potential for a decision support tool to reduce N2O emissions by modifying grazing management based on these parameters. Emission factors varied seasonally with the highest EFs in autumn and were also dependent on soil type, with the lowest EFs observed from well-drained and the highest from imperfectly drained soil. The EFs averaged 0.31 and 1.18% for cattle dung and urine, respectively, both of which were considerably lower than the IPCC default value of 2%. These results support both lowering and disaggregating EFs by excreta type.

The effect of nitrification inhibitors on nitrous oxide emissions from cattle urine depositions to grassland under summer conditions in the UK.

Nitrous oxide (N2O) has become the prime ozone depleting atmospheric emission and the third most important anthropogenic greenhouse gas, with a global warming potential approximately 300 times higher than CO2. Nitrification and denitrification are processes responsible for N2O emission from the soil after nitrogen input. The application of a nitrification inhibitor can reduce N2O emissions from these processes. The objective of this study was to assess the effect of two different nitrification inhibitors (dicyandiamide (DCD) and a commercial formulation containing two pyrazole derivatives (PD), 1H-1,2,4-triazole and 3-methylpyrazole) on N2O emissions from cattle urine applications for summer grazing conditions in the UK. Experiments were conducted under controlled conditions in a laboratory incubator and under field conditions on a grassland soil. The N2O emissions showed similar temporal dynamics in both experiments. DCD concentration in the soil showed an exponential degradation during the experiment, with a half-life of the order of only 10d (air temperature c. 15 °C). DCD (10 kg ha(-1)) and PD at the highest application rate (3.76 kg ha(-1)) reduced N2O emissions by 13% and 29% in the incubation experiment and by 33% and 6% in the field experiment, respectively, although these reductions were not statistically significant (P>0.05). Under UK summer grazing conditions, these nitrification inhibitors appear to be less effective at reducing N2O emissions than reported for other conditions elsewhere in the literature, presumably due to the higher soil temperature.

Mustard catch crop enhances denitrification in shallow groundwater beneath a spring barley field.

Over-winter green cover crops have been reported to increase dissolved organic carbon (DOC) concentrations in groundwater, which can be used as an energy source for denitrifiers. This study investigates the impact of a mustard catch crop on in situ denitrification and nitrous oxide (N2O) emissions from an aquifer overlain by arable land. Denitrification rates and N2O-N/(N2O-N+N2-N) mole fractions were measured in situ with a push-pull method in shallow groundwater under a spring barley system in experimental plots with and without a mustard cover crop. The results suggest that a mustard cover crop could substantially enhance reduction of groundwater nitrate NO3--N via denitrification without significantly increasing N2O emissions. Mean total denitrification (TDN) rates below mustard cover crop and no cover crop were 7.61 and 0.002 µg kg(-1) d(-1), respectively. Estimated N2O-N/(N2O-N+N2-N) ratios, being 0.001 and 1.0 below mustard cover crop and no cover crop respectively, indicate that denitrification below mustard cover crop reduces N2O to N2, unlike the plot with no cover crop. The observed enhanced denitrification under the mustard cover crop may result from the higher groundwater DOC under mustard cover crop (1.53 mg L(-1)) than no cover crop (0.90 mg L(-1)) being added by the root exudates and root masses of mustard. This study gives insights into the missing piece in agricultural nitrogen (N) balance and groundwater derived N2O emissions under arable land and thus helps minimise the uncertainty in agricultural N and N2O-N balances.

Slow delivery of a nitrification inhibitor (dicyandiamide) to soil using a biodegradable hydrogel of chitosan.

Using chemical inhibitors to reduce soil nitrification decreases emissions of environmental damaging nitrate and nitrous oxide and improves nitrogen use efficiency in agricultural systems. The efficacy of nitrification inhibitors such as dicyandiamide (DCD) is limited in soil due to biodegradation. This study investigated if the persistence of DCD could be sustained in soil by slow release from a chitosan hydrogel. DCD was encapsulated in glyoxal-crosslinked chitosan beads where excess glyoxal was (i) partly removed (C beads) or (ii) allowed to dry (CG beads). The beads were tested in water and in soil. The beads contained two fractions of DCD: one which was quickly released in water, and one which was not. A large DCD fraction within C beads was readily available: 84% of total DCD bead content was released after 9h immersion in water, while between 74% and 98% was released after 7d in soil under low to high moisture conditions. A lower percentage of encapsulated DCD was readily released from CG beads: 19% after 9h in water, and 33% after 7d in soil under high rainfall conditions. Kinetic analysis indicated that the release in water occurred by quasi-Fickian diffusion. The results also suggest that DCD release was controlled by bead erosion and the leaching of glyoxal derivatives, predominantly a glyoxal-DCD adduct whose release was positively correlated with that of DCD (R(2)=0.99, p⩽0.0001). Therefore, novel chitosan/glyoxal composite beads show a promising slow-release potential in soil for agrochemicals like DCD.

Evaluating the utility of 15N and 18O isotope abundance analyses to identify nitrate sources: A soil zone study.

(15)N and (18)O isotope abundance analyses in nitrate (NO(3)(-)) (expressed as δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) values respectively) have often been used in research to help identify NO(3)(-) sources in rural groundwater. However, questions have been raised over the limitations as overlaps in δ values may occur between N source types early in the leaching process. The aim of this study was to evaluate the utility of using stable isotopes for nitrate source tracking through the determination of δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) in the unsaturated zone from varying N source types (artificial fertiliser, dairy wastewater and cow slurry) and rates with contrasting isotopic compositions. Despite NO(3)(-) concentrations being often elevated, soil-water nitrate poorly mirrored the (15)N content of applied N and therefore, δ(15)N-NO(3)(-) values were of limited assistance in clearly associating nitrate leaching with N inputs. Results suggest that the mineralisation and the nitrification of soil organic N, stimulated by previous and current intensive management, masked the cause of leaching from the isotopic prospective. δ(18)O-NO(3)(-) was of little use, as most values were close to or within the range expected for nitrification regardless of the treatment, which was attributed to the remineralisation of nitrate assimilated by bacteria (mineralisation-immobilisation turnover or MIT) or plants. Only in limited circumstances (low fertiliser application rate in tillage) could direct leaching of synthetic nitrate fertiliser be identified (δ(15)N-NO(3)(-)<0‰ and δ(18)O-NO(3)(-)>15‰). Nevertheless, some useful differences emerged between treatments. δ(15)N-NO(3)(-) values were lower where artificial fertiliser was applied compared with the unfertilised controls and organic waste treatments. Importantly, δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) variables were negatively correlated in the artificial fertiliser treatment (0.001≤p≤0.05, attributed to the varying proportion of fertiliser-derived and synthetic nitrate being leached) while positively correlated in the dairy wastewater plots (p≤0.01, attributed to limited denitrification). These results suggest that it may be possible to distinguish some nitrate sources if analysing correlations between δ variables from the unsaturated zone. In grassland, the above correlations were related to N input rates, which partly controlled nitrate concentrations in the artificial fertiliser plots (high inputs translated into higher NO(3)(-) concentrations with an increasing proportion of fertiliser-derived and synthetic nitrate) and denitrification in the dairy wastewater plots (high inputs corresponded to more denitrification). As a consequence, nitrate source identification in grassland was more efficient at higher input rates due to differences in δ values widening between treatments.

Accumulation of the pro-apoptotic factor Bak is controlled by antagonist factor Mcl-1 availability.

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to reinforce or downgrade signals leading to the irrevocable entry into apoptosis. Bak (Bcl-2-antagonist killer) is a mitochondrial pore-forming pro-apoptotic effector inhibited through titration by the anti-apoptotic protein Mcl-1 (Myeloid cell leukemia-1). Viruses have taken advantage of proteasome-dependent degradation of Bak as a mechanism to prevent apoptosis in infected cells. It is not clear however whether regulation of Bak protein level is involved in other physiological processes. In this report, we show that Mcl-1 level is paralleled by Bak while a Mcl-1 non-interacting mutant of Bak does not accumulate in cells. This mechanism is proteasome independent. Following serum withdrawal, Bak accumulation becomes independent of Mcl-1 level and cells are sensitized to pro-apoptotic stimuli. Based on these results, we propose that regulation of Mcl-1-Bak steochiometry is a control mechanism used as a checkpoint to prevent or allow entry into apoptosis.

Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation.

Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor which is activated by hypoxia and involved in the adaptative response of the cell to oxygen deprivation. During hypoxic stress, HIF-1 triggers the overexpression of genes coding for glycolytic enzymes and angiogenic factors. To be active HIF-1 must be phosphorylated. HIF-1 is a substrate for various kinase pathways including PI-3K and the MAP kinases ERK and p38. Several transduction pathways have been proposed which act downstream of putative oxygen sensors and lead to the activation of these kinases. In this review, we summarize some of the latest advances describing the possible signaling pathways leading to HIF-1 phosphorylation and subsequent activation. The physiological relevance of these regulations is also discussed.

c-JUN gene induction and AP-1 activity is regulated by a JNK-dependent pathway in hypoxic HepG2 cells.

Hypoxia is an important pathophysiological stress that occurs during blood vessel injuries and tumor growth. It is now well documented that hypoxia leads to the activation of several transcription factors which participate in the adaptive response of the cells to hypoxia. Among these transcription factors, AP-1 is rapidly activated by hypoxia and triggers bFGF, VEGF, and tyrosine hydroxylase gene expression. However, the mechanisms of AP-1 activation by hypoxia are not well understood. In this report, we studied the events leading to AP-1 activation in hypoxia. We found that c-jun protein accumulates in hypoxic HepG2 cells. This overexpression is concomitant with c-jun phosphorylation and JNK activation. Moreover, we showed that AP-1 is transcriptionally active. We also observed that AP-1 transcriptional activity is inhibited by a MEKK1 dominant negative mutant. Moreover, the MEKK1 dominant negative mutant as well as deletion of the AP-1 binding sites within the c-jun promoter inhibited the c-jun promoter activation by hypoxia. All together, these results indicate that, in hypoxic HepG2 cells, AP-1 is activated through a JNK-dependent pathway and that it is involved in the regulation of the c-jun promoter, inducing a positive feedback loop on AP-1 activation via c-jun overexpression.

HIF-1 and AP-1 cooperate to increase gene expression in hypoxia: role of MAP kinases.

HIF-1 is the main transcription factor responsible for increased gene expression in hypoxia: VEGF, erythropoietin, GLUT-1, and glycolytic enzymes are such target genes and all participate in the adaptative response of cells to hypoxia. AP-1 activation by hypoxia has also been demonstrated in several cell lines and it cooperates with HIF-1 for increasing VEGF gene transcription in hypoxia. Both HIF-1 and AP-1 activation by hypoxia seems to involve members of the MAP kinase family. Here, we summarize the data indicating that ERK and JNK are needed for activation of HIF-1 and AP-1, respectively.

A model for the complex between the hypoxia-inducible factor-1 (HIF-1) and its consensus DNA sequence.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated by hypoxia. When activated, HIF-1 mediates the differential expression of genes such as erythropoietin and Vascular Endothelial Growth Factor (VEGF) during hypoxia. It is composed of two different subunits, HIF-1alpha and ARNT (Aryl Receptor Nuclear Translocator). These two subunits belong to the bHLH (basic Helix-Loop-Helix) PAS (Per, Ahr/ARNT, Sim) family. The bHLH domain of these factors is responsible for dimerization through the two helices and for DNA binding through their basic domain. In this work, we used various methods of molecular modeling in order to develop a 3D structure for the HIF-1 bHLH domain bound to its DNA consensus sequence. Firstly, the 3D structure of the bHLH domain of both subunits based on their amino acid sequence was defined. Secondly, we compared this model with data from known crystal structures of basic leucine zipper-DNA and bHLH-DNA complexes in order to determine a potential canvas for HIF-1. Thirdly, we performed a manual approach of the HIF-1 bHLH domain onto the DNA recognition site using this canvas. Finally, the protein-DNA complex 3D structure was optimized using a Monte Carlo program called MONTY. The model predicted a pattern of interactions between amino acids and DNA bases which reflect for ARNT what is experimentally observed among different X-ray structures of other bHLH transcription factors possessing the H (His), E (Glu), R (Arg) triad, as ARNT does. On the other hand, only the Arg residue is conserved in HIF- 1alpha. We propose from this model that a serine replaces the histidine while an alanine and a lysine also make contacts with DNA. From these results, we postulate that the specificity of HIF-1 toward its DNA sequence could be driven by the HIF-1alpha subunit. The predicted model will be verified by X-Ray currently ongoing.

Identification of hypoxia-responsive messengers expressed in human microvascular endothelial cells using differential display RT-PCR.

In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT-PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase-1, prostate tumor inducing factor-1, enolase-alpha and prothymosin-alpha were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia-responsive genes.

Role of HIF-1 as a transcription factor involved in embryonic development, cancer progression and apoptosis (review).

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor first identified as being activated by hypoxia but also in normoxic conditions by insulin and IGF-2. It is able to induce the expression of glycolytic genes and hence the ATP production, it also regulates the expression of the angiogenic factor VEGF and stimulates erythropoiesis via EPO production. HIF-1 is a protein necessary for the normal embryonic and cardiovascular system development, but seems to be also involved in cancer progression and apoptosis. Thus, it appears that HIF-1 plays a central role in normal cellular functions and in tissue metabolism but it is also involved in pathological evolutions raising its interest as a therapeutic target. In this review, we summarize the dual role of HIF-1 as a major component of the embryo development, as well as an element of tumor progression and of anoxia-induced apoptosis.

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.

Hypoxia-induced activation of HIF-1: role of HIF-1alpha-Hsp90 interaction.

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH-Per-aryl hydrocarbon nuclear translocator (ARNT)-Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), involved in the response to hypoxia, also belongs to the bHLH-PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF-1 activation by hypoxia. Using a EGFP-HIF-1alpha fusion protein, co-immunoprecipitation experiments evidenced that the chimeric protein expressed in COS-7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH-PAS domain of HIF-1alpha. Moreover, Hsp90 is not co-translocated with HIF-1alpha into the nucleus. At last, we showed that Hsp90 activity is essential for HIF-1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF-1alpha activation.

HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis elements located within the 5'UTR.

Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of two subunits, HIF-1alpha and ARNT, which is activated under hypoxia. HIF-1alpha mRNA is expressed constitutively in a wide variety of cell types, whereas in some others HIF1A gene expression is upregulated by hypoxia. In this report, we show that in endothelial cells (HMEC-1) the HIF-1alpha mRNA expression level is the same in both normoxia and hypoxia. Deletion analysis experiments of the HIF1A promoter showed that in hypoxia HIF1A gene expression is upregulated through a short sequence located next to the transcription initiation site. We also show that in hypoxia another sequence located upstream from the +1 initiation site plays an inhibitory role on HIF1A transcription in HMEC-1 but not in hepatoma cells and brings back this expression level to that observed in normoxia. Finally, we demonstrate that HIF1A gene transcription is dependent on Sp1 binding sites and that the 5'UTR sequence also contains other important cis-acting elements.