Time-dependent alterations of cerebral proteins following short-term normobaric hyperoxia

Sufficient oxygenation is indispensable for cognitive performance in mammals. In order to assure adequate oxygenation and to prevent hypoxia in medicine or aviation, different approaches of oxygen delivery are realized. With regard to hyperoxia, it is well known that it increases the risk of tissue toxicity and inflammation by generating radical oxygen species. However, this impact of hyperoxia on the expression of specific brain proteins has not been evaluated in detail yet. The present study analyzes time-dependent changes in protein expression in rat brain after a short-term exposure to normobaric hyperoxia. Thirty-six Wistar rats were randomly assigned to six different groups, three normobaric hyperoxia (NH) groups or three normobaric normoxia (NN) groups, each consisting of n = 6 animals. NH animals were exposed to 100% oxygen, NN rats to 21% oxygen, each group for 3 h. One group of NH and one group of NN were killed immediately after the 3 h, one group each after 3 days and one group each after 7 days. Rat brains were removed for analysis and whole brain detergent protein lysates were separated via two-dimensional gel electrophoresis followed by subsequent identification of protein expression alterations by peptide mass fingerprinting using mass spectrometry. Also, a functional network mapping and molecular pathway analysis were carried out. Statistical analysis was performed using analysis of variance (ANOVA) with Bonferroni correction using P < 0.01. Physiological parameters of the animals did not differ significantly between the two groups except for partial oxygen pressure (580 vs. 89 mmHg; P < 0.05). The expression of nine proteins was found to be significantly altered (five up-regulated: GOT1, CCT2, TCP1, G6PD, and ALB; four down-regulated: PEBP1, PRDX2, ENO1, and MDH1). IPA generated a network with eight focus proteins associated with pathways in “cell death, cancer, and signalling”. Although hyperoxia was normobaric and induced for only 3 h, significant changes in brain protein expression were detectable immediately after the 3 h, after 3 days, as well as after 7 days. This may indicate effects on brain protein expression take place in the rat brain following a relatively short period of hyperoxia.     

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia

This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO₂ = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.     

Influence of inorganic nitrogen sources on K+/Na+ homeostasis and salt tolerance in sorghum plants

This work aimed to study the regulation of K⁺/Na⁺ homeostasis and the physiological responses of salt-treated sorghum plants [Sorghum bicolor (L.) Moench] grown with different inorganic nitrogen (N) sources. Four days after sowing (DAS), the plants were transferred to complete nutrient solutions containing 0.75 mM K⁺ and 5 mM N, supplied as either NO₃ ^? or NH₄ ⁺. Twelve DAS, the plants were subjected to salt stress with 75 mM NaCl, which was applied in two doses of 37.5 mM. The plants were harvested on the third and seventh days after the exposure to NaCl. Under the salt stress conditions, the reduction of K⁺ concentrations in the shoot and roots was higher in the culture with NO₃ ^? than with NH₄ ⁺. However, the more conspicuous effect of N was on the Na⁺ accumulation, which was severely limited in the presence of NH₄ ⁺. This ionic regulation had a positive influence on the K⁺/Na⁺ ratio and the selective absorption and transport of K⁺ in the plants grown with NH₄ ⁺. Under control and salt stress conditions, higher accumulation of free amino acids and soluble proteins was promoted in NH₄ ⁺ grown roots than NO₃ ^? grown roots at both harvesting time, whereas higher accumulation of soluble sugars was observed only at 7 days of salt stress exposure. Unlike the NH₄ ⁺ grown plants, the gas exchanges of the NO₃ ^? grown plants were reduced after 7 days of salt stress. These results suggest that external NH₄ ⁺ may limit Na⁺ accumulation in sorghum, which could contribute to improving its physiological and metabolic responses to salt stress.     

Hyperoxia increases oxygen radical production in rat lung homogenates

Lung damage during hyperoxia has been postulated to be due to increased rates of local organ oxygen radical production. Lung homogenate respiration was inhibited with cyanide, and residual respiration was used as an indicator of electron diversion to O₂^? and H₂O₂. Cyanide-resistant respiration in lung homogenates, supplemented with 1 mm NADH, increased linearly with oxygen tension, and accounted for 7% of total respiration in air and for 17% of total respiration when homogenates were incubated in 80% oxygen. Exposure of rats to 85% oxygen for 7 days induces tolerance to the lethal effects of 100% oxygen. Rats which previously breathed 85% oxygen for 7 days had a greater CN^?-resistant respiration than control rats. This implies that adaptation to hyperoxia does not include decreased lung tissue oxygen radical production as indicated by CN^?-resistant respiration. One possible explanation for the increased CN^?-resistant respiration in oxygen tolerant rat lungs is that they contain increased cell mass. Lung homogenates of rats exposed to 85% oxygen for 7 days also had 2.5 times greater thiobarbituric acid positive material than controls, indicating that increased lung lipid peroxidation occurs as a consequence of hyperoxia. Incubation of normal rat lung homogenates under hyperoxic conditions also acutely increased lipid peroxidation, which could be inhibited by both superoxide dismutase and catalase. This confirms that hyperoxia enhances cellular production of O₂^? and H₂O₂ and implies an essential role for both O₂^? and H₂O₂ in hyperoxic lung damage.     

Effect of intermittent hypoxia or hyperoxia on lung development in preterm rat neonates during constant oxygen therapy

Impaired lung development is a major negative factor in the survival of preterm neonates. The present study was aimed to investigate the impact of constant oxygen, intermittent hyperoxia, and hypoxia on the lung development in preterm rat neonates. Neonatal rats were exposed to 40% O₂ with or without brief hyperoxia episodes (95% O₂) or brief hypoxia episodes (10% O₂) from day 0 to day 14, or to room air. The body weight, radical alveolar count (RAC), and total antioxidant capacity (TAOC) were significantly lower whereas the lung coefficient and malondialdehyde (MDA) were significantly higher in the hyperoxia and hypoxia groups than the air control and constant oxygen group at day 7, day 14, and day 21 after birth. The lung function indexes were reduced by intermittent hyperoxia and hypoxia. In contrast, the constant oxygen therapy increased the lung function. HIF-1α and VEGF expression were significantly increased by hypoxia and decreased by hyperoxia. The constant oxygen therapy only decreased the HIF-1α expression at day 14 and 21. In summary, the constant oxygen treatment promoted lung function without affecting the antioxidative capacity in preterm rat neonates. While intermittent hyperoxia and hypoxia inhibited lung development, decreased antioxidative capacity, and dysregulated HIF-1α/VEGF signaling in preterm rat neonates.     

The apoptosis of peripheral blood lymphocytes promoted by hyperbaric oxygen treatment contributes to attenuate the severity of early stage acute pancreatitis in rats

The aim of this study was to investigate the immunoregulatory effects of hyperbaric oxygen (HBO) via promoting the apoptosis of peripheral blood lymphocytes (PBLs) to attenuate the severity of early stage acute pancreatitis (AP) in rats. Additionally, the persistence of the HBO treatment effects was evaluated. One hundred and twenty male Wistar rats were randomized into four groups: sham, AP, AP + normobaric oxygen (NBO), and AP + HBO. Each group consisted of 30 rats. Four hours after the induction of AP, the 30 rats in the AP + NBO group were given normobaric oxygen treatment with 100 % oxygen at 1 atm for 90 min. The 30 rats in the AP + HBO group received 100 % oxygen at 2.5 atm for 90 min, with a compression/decompression time of 15 min. The 30 rats in the AP group remained untreated. At 6, 12, and 24 h after the induction of AP, surviving rats from each group were sacrificed, and the blood and tissue samples were collected for the following measurements: the partial pressure of oxygen (PaO₂) and oxygen saturation (SaO₂) of the arterial blood, the levels of serum amylase, lipase, interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-10 (IL-10), hepatocyte growth factor (HGF), and reactive oxygen species (ROS), and the mitochondrial membrane potential (?Ψm) of the PBLs. The expression levels of procaspase-3, caspase-3, procaspase-9, and caspase-9 were also evaluated in the PBLs. Additionally, the apoptosis of PBLs was assessed, and the pancreatic tissues were subjected to a histopathological analysis by pathological grading and scoring. The histopathology of the lung, liver, kidney, duodenum, and heart was also analyzed at 12 h after the induction of AP. Significant differences were found at 6 and 12 h after AP induction. The HBO treatment significantly elevated the PaO₂ and SaO₂ levels, and the ROS levels in the PBLs. Additionally, HBO downregulated the levels of amylase and lipase. The HBO treatment also reduced the ?Ψm levels, upregulated the expression of caspase-3 and caspase-9, and increased the apoptosis rate of the PBLs. Moreover, the HBO treatment decreased the serum concentrations of IL-2, IFN-γ and HGF, and reduced the pathological scores of the pancreatic tissue. The histopathological changes of the lung, liver, kidney, duodenum, and heart were also improved. A significant elevation of IL-10 occurred only at the 12-h time point. However, no obvious differences were found at the 24-h time point. This study demonstrated that the HBO treatment can promote the apoptosis of PBLs via a mitochondrial-dependent pathway and inhibit the inflammatory response. These immunoregulatory effects may play an important therapeutic role in attenuating the severity of early stage AP. The repeated administration of HBO or the use of HBO in combination with other approaches may further improve outcomes.     

Modulation of osmotic adjustment and antioxidant status in salt-stressed leaves of Thermopsis turcica

Thermopsis turcica is distributed naturally in saline soils. Interestingly, how T. turcica can live in harsh salt conditions is unknown. To study its defense responses under salinity, T. turcica was grown in a medium containing 100 and 200 mM NaCl for 7 and 14 days. Physiological parameters, ion contents, reactive oxygen species accumulation, activities of antioxidant enzymes/isozymes, NADPH oxidase enzyme/isozyme, lipid peroxidation (TBARS) and osmolyte contents were investigated. Stress caused a rapid decline in relative growth rate, relative water content and chlorophyll fluorescence (F _v/F _m) under both NaCl treatments. These traits were more suppressed at 200 mM NaCl. The decline in osmotic potential (Ψ _Π) with salinity increased the gradient for water flux into the cell and assisted in turgor maintenance. The increased membrane permeability under stress caused the entrance of excess Na⁺ and K⁺ into the cell. Stress decreased superoxide dismutase, catalase and peroxidase after 14 days of growth in 200 mM NaCl, whereas glutathione reductase (GR) increased throughout the experiment. While ascorbate peroxidase (APX) increased by 44 % at 7 days, it decreased after 14 days exposure to 200 mM NaCl. 200 mM NaCl caused the highest increase in TBARS at 14 days, indicating a decrease in OH^· scavenging activity. Increasing concentrations of salinity caused an increase in glycine betaine (GB) and choline (Cho), though an increase in proline was only observed at 200 mM NaCl for 14 days. Briefly, H₂O₂ was more efficiently eliminated in 100 mM-treated plants by the ascorbate–glutathione cycle in which APX acts a strong catalyst together with GR. Also, Cho and GB help to maintain osmotic adjustment and cytoplasmic function.     

Expression of autotaxin and lysophosphatidic acid receptors 1 and 3 in the developing rat lung and in response to hyperoxia

Abstract

We sought to evaluate lysophosphatidic acid (LPA) signaling improvement in lung development by assessing the expression of autotaxin and LPA receptor 1 and 3 (LPAR1 and LPAR3) in the neonatal rat lung during normal perinatal development and in response to hyperoxia. In the developmental study, rats were sacrificed on days 17, 19, and 21 of gestation; on postnatal days 1, 4, and 7; and at adulthood (postnatal 9 weeks). In the hyperoxia study, 42 postnatal 4-day-old rat pups were divided into seven groups and exposed to either 85% O₂ for 24, 72, or 120 h or room air for 0, 24, 72, or 120 h. The rats were then euthanized after 0, 24, 72, and 120 h of exposure. Immunofluorescence demonstrated that autotaxin, LPAR1, and LPAR3 proteins were broadly colocalized in airway epithelial cells, but mainly distributed in vascular endothelial and mesenchymal cells during the first postnatal week. The expression of autotaxin, LPAR1, and LPAR3 were increased during late gestation and then decreased after birth. Autotaxin expression and enzymatic activity were significantly increased at 72 and 120 h after exposure to hyperoxia. LPAR1 and LPAR3 expression was also increased after 120 h of hyperoxic exposure. These findings suggest that LPA-associated molecules were upregulated at birth and induced by hyperoxia in the developing rat lung. Therefore, the LPA pathway may be involved in normal lung development, including vascular development, as well as wound-healing processes of injured neonatal lung tissue, which is at risk of neonatal hyperoxic acute lung injury.     

Effect of gibberellic acid on the cyanobacterium <Emphasis Type="Italic">Nostoc linckia</Emphasis>

We investigated the influence of gibberellic acid (GA₃; 0, 1, 10, and 100 μM) on Nostoc linckia culture at 7, 14, and 21 days. The fresh and dry weight of N. linckia was increased considerably by the 10 and 100 μM GA₃ treatments. A reduction in heterocyst frequency was observed in cultures treated with 1 and 10 μM GA₃. Adding GA₃ to N. linckia culture had a little effect on cell size. The amount of chlorophyll a and carotenoids decreased at all concentrations of GA₃. The amount of phycocyanin increased up to twofold in 7-day-old culture treated with 1 μM GA₃, and similar changes were observed for allophycocyanin and phycoerythrin content after 7 days. The effect of GA₃ on reducing sugar content was different and was dependent on the growth period. A reduction in soluble sugar content was detected after GA₃ application in 7- and 14-day-old cyanobacteria. Cultures treated with GA₃ had a higher protein content after 14 days and a lower protein content after 7 and 21 days, and reduced nitrogenase activity after 7, 14, and 21 days. Our data show that GA₃ application can be a suitable and inexpensive way to increase N. linckia biomass and phycobiliprotein production.     

Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice

Background

Preterm newborns that receive oxygen therapy often develop bronchopulmonary dysplasia (BPD), which is abnormal lung development characterized by impaired alveologenesis. Oxygen-mediated injury is thought to disrupt normal lung growth and development. However, the mechanism of hyperoxia-induced BPD has not been extensively investigated. We established a neonatal mouse model to investigate the effects of normobaric hyperoxia on retinoid metabolism and retinoid receptor expression.

Methods

Newborn mice were exposed to hyperoxic or normoxic conditions for 15 days. The concentration of retinol and retinyl palmitate in the lung was measured by HPLC to gauge retinoid metabolism. Retinoid receptor mRNA levels were assessed by real-time PCR. Proliferation and retinoid receptor expression in A549 cells were assessed in the presence and absence of exogenous vitamin A.

Results

Hyperoxia significantly reduced the body and lung weight of neonatal mice. Hyperoxia also downregulated expression of RARα, RARγ, and RXRγ in the lungs of neonatal mice. In vitro, hyperoxia inhibited proliferation and expression of retinoid receptors in A549 cells.

Conclusion

Hyperoxia disrupted retinoid receptor expression in neonatal mice.     

The potential effect of sustained hypoxia on nitrogen cycling in sediment from the southern North Sea: a mesocosm experiment

The potential effect of sustained hypoxia (up to 70 days) on the production of N₂ gas through denitrification and anammox, as well as sediment–water exchange of nitrite, nitrate and ammonia, oxygen consumption and penetration, were measured in mesocosms using sediment collected from the southern North Sea (north of Dogger Bank). As expected, both the penetration of oxygen into, and consumption of oxygen by, the sediment decreased by 42 and 46 %, respectively, once hypoxia was established. Importantly, the oxygen regime did not change significantly (P > 0.05) during the experiment, suggesting that organic carbon was not depleted. During the first 10 days, the exchange of NO₃ ^?, NO₂ ^? and NH₄ ⁺ between the sediment and water was erratic but once a steady state was established the sediment acted as either a sink for fixed nitrogen under hypoxia or as a source in the controls. Over the course of the mesocosm experiment the rate of both anammox and denitrification increased, with anammox increasing disproportionately under hypoxia relative to the controls, whereas the rate of increase in denitrification was the same for both. Under sustained hypoxia the production of N₂ gas increased by 72 % relative to the controls, with this increase in N₂ production remaining constant regardless of the duration of hypoxia. Longer periods of stratification and oxygen depletion are predicted to occur more regularly in the bottom waters of shallow coastal seas as one manifestation of climate change. Under sustained hypoxia the potential for nitrogen removal by the production of N₂ gas in this region of the southern North Sea was estimated to increase from 2.1 kt N 150 days^?1 to 3.6 kt 150 days^?1, while the efflux of dissolved inorganic nitrogen ceased altogether; both of which could down regulate the productivity of this region as a whole.     

Synergistic protection against hyperoxia-induced lung injury by neutrophils blockade and EC-SOD overexpression

Background

Oxygen may damage the lung directly via generation of reactive oxygen species (ROS) or indirectly via the recruitment of inflammatory cells, especially neutrophils. Overexpression of extracellular superoxide dismutase (EC-SOD) has been shown to protect the lung against hyperoxia in the newborn mouse model. The CXC-chemokine receptor antagonist (Antileukinate) successfully inhibits neutrophil influx into the lung following a variety of pulmonary insults. In this study, we tested the hypothesis that the combined strategy of overexpression of EC-SOD and inhibiting neutrophil influx would reduce the inflammatory response and oxidative stress in the lung after acute hyperoxic exposure more efficiently than either single intervention.

Methods

Neonate transgenic (Tg) (with an extra copy of hEC-SOD) and wild type (WT) were exposed to acute hyperoxia (95% FiO₂ for 7 days) and compared to matched room air groups. Inflammatory markers (myeloperoxidase, albumin, number of inflammatory cells), oxidative markers (8-isoprostane, ratio of reduced/oxidized glutathione), and histopathology were examined in groups exposed to room air or hyperoxia. During the exposure, some mice received a daily intraperitoneal injection of Antileukinate.

Results

Antileukinate-treated Tg mice had significantly decreased pulmonary inflammation and oxidative stress compared to Antileukinate-treated WT mice (p < 0.05) or Antileukinate-non-treated Tg mice (p < 0.05).

Conclusion

Combined strategy of EC-SOD and neutrophil influx blockade may have a therapeutic benefit in protecting the lung against acute hyperoxic injury.     

Surfactant metabolism and anti-oxidative capacity in hyperoxic neonatal rat lungs: effects of keratinocyte growth factor on gene expression in vivo

Development of preterm infant lungs is frequently impaired resulting in bronchopulmoary dysplasia (BPD). BPD results from interruption of physiologic anabolic intrauterine conditions, the inflammatory basis and therapeutic consequences of premature delivery, including increased oxygen supply for air breathing. The latter requires surfactant, produced by alveolar type II (AT II) cells to lower surface tension at the pulmonary air:liquid interface. Its main components are specific phosphatidylcholine (PC) species including dipalmitoyl-PC, anionic phospholipids and surfactant proteins. Local antioxidative enzymes are essential to cope with the pro-inflammatory side effects of normal alveolar oxygen pressures. However, respiratory insufficiency frequently requires increased oxygen supply. To cope with the injurious effects of hyperoxia to epithelia, recombinant human keratinocyte growth factor (rhKGF) was proposed as a surfactant stimulating, non-catabolic and epithelial-protective therapeutic. The aim of the present study was to examine the qualification of rhKGF to improve expression parameters of lung maturity in newborn rats under hyperoxic conditions (85 % O₂ for 7 days). In response to rhKGF proliferating cell nuclear antigen mRNA, as a feature of stimulated proliferation, was elevated. Similarly, the expressions of ATP-binding cassette protein A3 gene, a differentiation marker of AT II cells and of peroxiredoxin 6, thioredoxin and thioredoxin reductase, three genes involved in oxygen radical protection were increased. Furthermore, mRNA levels of acyl-coA:lysophosphatidylcholine acyltransferase 1, catalyzing dipalmitoyl-PC synthesis by acyl remodeling, and adipose triglyceride lipase, considered as responsible for fatty acid supply for surfactant PC synthesis, were elevated. These results, together with a considerable body of other confirmative evidence, suggest that rhKGF should be developed into a therapeutic option to treat preterm infants at risk for impaired lung development.     

Dynamin 2 and c-Abl Are Novel Regulators of Hyperoxia-mediated NADPH Oxidase Activation and Reactive Oxygen Species Production in Caveolin-enriched Microdomains of the Endothelium

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47^phox, to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47^phox and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47^phox recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47^phox/dynamin 2 association (change in the dissociation constant (K_d) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47^phox, and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47^phox to CEMs and subsequent ROS production in lung endothelium.     

Three years of soil respiration in a mature eucalypt woodland exposed to atmospheric CO<Subscript>2</Subscript> enrichment

Large amounts of atmospheric N deposition cause negative effects on ecosystems. Effective mitigation strategies require the sources of N deposition to be identified and the contributions from individual sources to be quantified. Determination of the isotopic composition represents a useful approach in source apportionment. In this study, the δ¹⁵N-NH_x of wet and dry atmospheric deposition and the main NH₃ emission sources were analyzed at an urban, a suburban and a rural site in the Taihu Lake region of China. The 2-year average δ¹⁵N-\( {\text{NH}}_{4}^{ + } \) of precipitation was ? 3.0 ± 2.3, ? 3.1 ± 2.8 and ? 0.5 ± 2.8‰ for the urban, suburban and rural sites, respectively. These values were much lower than the corresponding values for particulate \( {\text{NH}}_{4}^{ + } \) (15.9, 15.2 and 14.3‰ at the urban, suburban and rural sites, respectively), and much higher than those of gaseous δ¹⁵N-NH₃ (? 16.7, ? 18.2 and ? 17.4‰ at the urban, suburban and rural sites, respectively). The δ¹⁵N-NH₃ of NH₃ from the main emission sources ranged from ? 30.8 to ? 3.3‰ for volatilized fertilizer, from ? 35.1 to ? 10.5‰ for emissions from a pig farm, and ? 24.7 to ? 11.3‰ for emissions from a dairy farm. Temporal variations of deposition δ¹⁵N-NH_x indicated that δ¹⁵N-NH_x values were lower in summer and autumn, but higher in winter and spring for both precipitation \( {\text{NH}}_{4}^{ + } \)-N and gaseous NH₃-N. Weather conditions such as temperature and precipitation significantly influenced the spatial and temporal distribution of isotope values of the deposition. Analysis of δ¹⁵N-NH_x in deposition and emission sources identified volatilized fertilizer and livestock wastes as the origins of both gaseous NH₃-N and precipitation \( {\text{NH}}_{4}^{ + } \)-N over the region. A stable isotope mixing model estimated that volatilized fertilizer and animal excreta contributed more than 65% to precipitation \( {\text{NH}}_{4}^{ + } \)-N, more than 60% to particulate \( {\text{NH}}_{4}^{ + } \)-N, and more than 75% to gaseous NH₃-N.     

Changes in Ecosystem Function Across Sedimentary Gradients in Estuaries

The input of terrestrial silt and clay (hereafter mud) into coastal environments can alter sediment grain size distribution affecting the structure and functioning of benthic communities. The relationship between sediment mud content and macrofaunal community structure has been well documented, but not the effects on ecosystem function. In 143 plots from the mid-intertidal sites in 9 estuaries, we measured sediment properties, macrofaunal community composition and fluxes of O₂ and NH₄ ⁺ across the sediment–water interface to derive process-based measures of ecosystem function across the sand–mud gradient. We observed reductions in measures of macrofaunal diversity and decreases in the maximum density of key bioturbating bivalves (Austrovenus stutchburyi and Macomona liliana) with increased mud content. Concurrently, the maximum rates of sediment oxygen consumption (SOC), NH₄ ⁺ efflux (NH₄ ⁺) and biomass standardized gross primary production (GPP_Chl-a ) also decreased with increasing mud content. Environmental predictors explained 34–39% (P = 0.005–0.01) of the total variation in ecosystem function in distance-based linear models. After partitioning out the effect of mud, A. stutchburyi abundance was positively correlated and explained 25 and 23% (P = 0.0001) of the variation of SOC and NH₄ ⁺, respectively. Also, mud content (negatively correlated) and temperature (positively correlated) explained 26% of variability in GPP_Chl-a (P = 0.0001). Our results highlight the importance of increased mud content and the associated reduction in the abundance of strongly interacting key species on the loss of ecosystem function in intertidal sand flats.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.