Uptake of ammonium and nitrate ions from acid mist applied to Sitka spruce [Picea sitchensis (Bong.) Carr.] grafts over the course of one growing season

Cloned Sitka spruce grafts were subjected to NH₄NO₃ mist (1 mm day^-1 equivalent) acidified with H₂SO₄, for 4 days a week, from April until November. Three N treatments at pH 5.0 used 0.01 mol m^{-3 15}NH₄ ¹⁵NH₃, 1.6 mol m^{-3 15}NH₄ NO₃ or 1.6 mol m^-3 NH₄ ¹⁵NO₃. At pH 2.5, the treatments were¹⁵NH₄ ¹⁵NO₃,¹⁵NH₄NO₃ and NH₄ ¹⁵NO₃ all at 1.6 mol m^-3. At the end of the growing season,¹⁵N was found in every part of the trees, even when NH₄NO₃ was supplied at 0.01 mol m^-3. It was shown that both and could enter needles to a similar degree and be transported to the roots. No differences between pH treatments or clones could be demonstrated. Entry of labelled N via the roots, in those trees with unprotected compost, was reflected in higher fine root¹⁵N content, but no change in shoot¹⁵N levels. Per cent incorporation expressed as labelled N as a percentage of total N, was generally at its highest in bark, and in older needles in all treatments, values ranging from 5 to 11%. When¹⁵N content was expressed as total content for each tree part on a per tree basis, 25–36% was found in current year needles, with a further 12% in current year bark. The most reproducible data was that for dry weight of tree parts as per cent of the whole tree, where proportions compared closely between treatments and the two clones used. The implications of the results for cuticular transport mechanisms, N storage and internal cycling are discussed.     

Improved lineshape and sensitivity in the HNCO-family of triple resonance experiments

A simple scheme is presented for the suppression of dispersive contributions to cross peaks in HNCO-type spectra where the ¹⁵N chemical shift is recorded in a constant-time manner immediately prior to the transfer from ¹⁵ N to ¹HN at the end of the sequence. These dispersive contributions arise when the delay for refocusing the ¹⁵N-¹³CO one-bond coupling is set to less than 0.5/¹J_N,CO and when ² J_HN,CO 0. Improvements in sensitivity in ¹HN detected experiments recorded on ¹⁵ N,¹³C-labeled samples can be realized by application of ¹³CO/¹³ decoupling during acquisition. Sensitivity gains on the order of 15% and 5% have been obtained for an SH3 domain (62 residues) and maltose binding protein (370 residues), respectively.     

EPR evidence for generation of hydroxyl radical triggered byN-acetylchitooligosaccharide elicitor and a protein phosphatase inhibitor in suspension-cultured rice cells

Summary N-acetylchitooligosaccharides, fragments of the backbone of fungal cell wall, trigger rapid membrane responses such as transient depolarization, and elicit defense reactions including phytoalexin production in suspension-cultured rice cells. The generation of reactive oxygen species triggered by the oligosaccharide signal was analyzed with EPR spectroscopy using a spin trapping system, 4-pyridyl 1-oxideN-tert-butyl nitrone (4-POBN) and ethanol. OH generation was detected as the -hydroxyethyl adduct of 4-POBN after elicitation. Superoxide dismutase, catalase or diethylenetriamine pentaacetic acid, a metal chelator, inhibited generation, proposing the following reaction sequence: generation of in response to the oligosaccharide elicitor, followed by dismutation to H₂O₂, then generation of by the reaction of H₂O₂ with Fe²⁺ that is generated by the reduction of Fe³⁺ by . Generation of the same reactive oxygen species was also triggered by calyculin A, a protein phosphatase inhibitor, alone, suggesting the involvement of protein phosphorylation in its regulation during the oligosaccharide signal transduction.Abbreviations DMPO 5,5-dimethyl-1-pyrroline N-oxide - DTPA diethylenetriamine pentaacetic acid - 4-POBN 4-pyridyl 1-oxideN-tert-butylnitrone - SOD Superoxide dismutase - 4-hydroxy-TEMPO 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl     

Monovalent cation permeabilities of the potassium systems in the crab giant axon

Summary Permeability ratios for pairs of monovalent cations permeating the two potassium systems proposed for the giant axon of the crabCarcinus maenas (M. E. Quinta-Ferreira, E. Rojas & N. Arispe,J. Membrane Biol. 66:171–181, 1982b) were estimated from measurements of the reversal potential of the currents under voltage-clamp conditions. With K⁺ inside the axon, permeability ratios from the reversal potential of the currents through the late channel are:P _Rb/P _K=0.9, /P _K<0.2 andP _Cs/P _K=0.18. With Cs⁺ inside the ratios are:P _K/P _Cs=8.7,P _Rb/P _Cs=7.1 and /P _Cs=2.4. The analysis of the inward currents carried by Rb⁺ or NH ₄ ⁺ showed similar reversal potentials for the early transient component and the late sustained component. Whence, the sequence of permeabilities for the two types of potassium channels is:P _K>P _Rb> >P _Na=P _Cs. The time constants for the activation of the two components recorded either in K-, Rb-, or NH₄-artificial seawater are twice as large as the corresponding time constants measured in Na-artificial seawater.     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: Application to <Superscript>15</Superscript>N relaxation measurements

A quantitative analysis of 2D ¹H-¹⁵N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of ¹⁵N T₁ relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D ¹H-¹⁵N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied.     

Cardiovascular responses to facial cooling during low and moderate intensity exercise

To investigate the hypothesis that facial cooling (FC) exerts a greater influence on the cardiovascular system at lower versus higher levels of exercise, this study examined the effect of facial cooling [mean (SE): 0 (2)°C at 0.8 m·s^–1 wind velocity] during 30 min low [35% maximum oxygen consumption ( O_2max)] and moderate (70% O_2max) levels of cycle ergometry in the supine position. Five male subjects were assigned in random order to four exercise conditions: (1) FC at 35% O_2max(FC35), (2) no cooling (NFC35), (3) FC at 70% O_2max(FC70), and (4) no cooling (NFC70). Heart rate (f _c), stroke volume (V _s), and cardiac output ( _c) were measured at rest and every 10 min of exercise using impedance cardiography. During FC35, the change in f _c [mean (SE)] was significantly lower (P < 0.05) than NFC35 at 10 [22 (5) vs 31 (3) beats· min^–1], 20 [29 (6) vs 35 (3) beats·min^–1], and 30 [29 (5) vs 38 (4) beats·min^–1] min. No differences in f _c were observed between FC70 and NFC70. Furthermore, FC had no effect on V _s or _cat either exercise intensity. However, when comparing the FC70 and NFC70 conditions, there was a significant main effect (P<0.05) in mean arterial pressure (P _a) response with cooling despite the fact that neither V _s or _cwere different from the NFC70 control. The increase (P < 0.05) in the estimated change in systemic vascular resistance ( _a· _c ^–1) could partly explain the relative rise in _aat FC70. No pressor effect of cooling was observed at 35% O_2max. The results suggest that the FC condition promotes exercise bradycardia at low levels of exercise and exerts a greater pressor response during moderate exercise.     

Accurate measurement of <Superscript>15</Superscript>N–<Superscript>13</Superscript>C residual dipolar couplings in nucleic acids

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ ¹H–¹³C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H₁–C₁–N_1/9 (MQ-HCN-QJ) or H_6/8–C_6/8–N_1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2J_NC) (attenuated spectrum). The different types of ¹⁵N–¹³C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time ¹⁵N evolution period and the offset of two frequency-selective ¹³C pulses. The methods are demonstrated for a uniformly ¹³C, ¹⁵N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for ¹D_NC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including ¹³C–¹H, ¹³C–¹³C and ¹H–¹H dipolar couplings.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0646-2.     

Inorganic nitrogen source utilization byFagus crenata on different soil types

The nitrogen source utilization by Fagus crenata distributed on soils with different forms of inorganic nitrogen in a cool-temperate deciduous forest in central Japan was determined by measuring foliar ¹⁵N. Two soil habitat types along a slope were delineated based on nitrogen transformation patterns, i.e., soils with high net nitrification rates and with no or low net nitrification, respectively. Despite differences in soil types, the study species, F. crenata, was distributed along the entire slope. The foliar ¹⁵N value of F. crenata from the lower slope area was significantly lower than that from the upper slope. Given the finding of a previous study that the ¹⁵N of NO₃^– was lower than that of NH₄⁺, our results indicate that reliance on NO₃^– as a nitrogen source was greater in the lower slope area than in the upper slope area. Differences in the values of foliar ¹⁵N were about 1, which is far less than the 10 ¹⁵N value of soil inorganic N reported in the previous study. This discrepancy might suggest that the study species utilized NO₃^– even in the upper site where net nitrification had not been detected. Measurements of nitrate reductase activity, an index of NO₃^– uptake, also supported this interpretation. Nitrate reduction occurred in leaves and roots at both the lower and the upper sites. Thus, the study species may be able to use NO₃^– even in soils with no net nitrification, a factor that could allow the distribution of F. crenata along the entire length of the slope.     

Characterization of microbial NO production, N2O production and CH4 oxidation initiated by aeration of anoxic rice field soil

Intermittent drainage of rice fields isdiscussed as an option to mitigate emission ofCH₄, an important greenhouse gas. HoweverN₂O, a potentially more effective greenhouse gas,may be emitted during the aeration phase. Therefore,the metabolism of NO, N₂O, NH ,NO and NO and the kinetics ofCH₄ oxidation were measured after aeration ofmethanogenic rice field soil. Before aeration, thesoil contained NH in relatively highconcentrations (about 4 mM), while NO andNO were almost undetectable. Immediatelyafter aeration both NO and N₂O were produced withrates of about 15 pmol h^-1 gdw^-1 and 5 pmolh^-1 gdw^-1, respectively. Simultaneously,NH decreased while NO accumulated. Later on, NO was depletedwhile NO concentrations increased.Characteristic phases of nitrogen turnover wereassociated with the activities of ammonium oxidizers,nitrite oxidizers and denitrifiers. Oxidation ofNH and production of NO and N₂O wereinhibited by 10 Pa acetylene demonstrating thatnitrification was obligatory for the initiation ofnitrogen turnover and production of NO and N₂O.Ammonium oxidation was not limited by the availableNH and thus, concomittant production of NOand N₂O was not stimulated by addition ofNH . However, addition of NO stimulated production of NO and N₂O in bothanoxic and aerated rice soil slurries. In this case,10 Pa acetylene did not inhibit the production of NOand N₂O demonstrating that it was due todenitrification which was obviously limited by theavailability of NO . In the aerated soilslurries CH₄ was only oxidized if present atelevated concentrations >50 ppmv CH₄). Atatmospheric CH₄ concentrations (1.7 ppmv)CH₄ was not consumed, but was even slightly produced.CH₄ oxidation activity increased afterpreincubation at 20% CH₄, and then CH₄was also oxidized at atmospheric concentrations. CH₄oxidation kinetics exhibited sigmoid characteristicsat low CH₄ concentrations presumably because ofinhibition of CH₄ oxidation by NH .     

Hydrogen-isotope composition of leaf water in C3 and C4 plants: its relationship to the hydrogen-isotope composition of dry matter

The natural abundance hydrogen-isotope composition of leaf water ( ) and leaf organic matter ( _D ^org ) was measured in leaves of C₃ and C₄ dicotyledons and monocotyledons. The value of leaf water showed a marked diurnal variation, greatest enrichment being observed about midday. However, this variation was greater in the more slowly transpiring C₄ plants than in C₃ plants under comparable environmental conditions. A model based on analogies with a constant feed pan of evaporating water was developed and the difference between C₃ and C₄ plants expressed in terms of either differences in kinetic enrichment or different leaf morphology. Microclimatic and morphological features of the leaves which may be associated with this factor are discussed. There was no daily excursion in the _D ^org value in leaves of either C₃ or C₄ plants. When _D ^org values were referenced to the mean values during the period of active photosynthesis, the discrimination against deuterium during photosynthetic metabolism (D) was greater in C₃ plants (-117 to -121) than in C₄ plants (-86 to -109).These results show that the different water use strategies of C₃ and C₄ plants are responsible for the measured difference in deuterium-isotope composition of leaf water. However, it is unlikely that these physical processes account fully for the differences in hydrogen-isotope composition of the products of C₃ and C₄ photosynthetic metabolism.Symbols Hydrogen-isotope composition of leaf water - _D ^org hydrogen-isotope composition of leaf organic matter     

Times to exhaustion at 100% of velocity at [(V)\dot]\textO\text2 \dot V{\text{O}}_{\text{2}} max and modelling of the time-limit / velocity relationship in elite long-distance runners

The aim of this study was to measure running times to exhaustion (Tlim) on a treadmill at 100% of the minimum velocity which elicits _{max max} in 38 elite male long - distance runners _max = 71.4 ± 5.5 ml.kg^–1.min^–1 and _max = 21.8 ± 1.2 km.h^–1). The lactate threshold (LT) was defined as a starting point of accelerated lactate accumulation around 4 mM and was expressed in _max. Tlim value was negatively correlated with _max (r = -0.362, p< 0.05) and _max (r = –0.347, p< 0.05) but positively with LT (%v _max) (r = 0.378, p < 0.05). These data demonstrate that running time to exhaustion at _max in a homogeneous group of elite male long-distance runners was inversely related to _max and experimentally illustrates the model of Monod and Scherrer regarding the time limit-velocity relationship adapted from local exercise for running by Hughson et al. (1984) .     

Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo^–) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 M) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 M and 2 M resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the variable part of chlorophyll fluorescence (which is the recombination luminescence of the pair P₆₈₀ ⁺ Pheo^–). A 50% inhibition was induced by 5 M K-15 and the effect was maximal in the range 20 to 200 M. Photooxidation of P₆₈₀ in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 M). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo^– and P₆₈₀ ⁺ (or Z⁺) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for Q_A missing in the preparation.At a concentration of 0.5–50 M K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl₂. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo^– to cytochrome b559.Abbreviations K-15 4-[methoxy-bis-(trifluoromethyl)methyl]-2,6-dinitrophenyl hydrazone methyl ketone - P₆₈₀ the primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A and Q_B the primary and the secondary electron acceptor of PS II - RC reaction centre - SiMo silicomolybdate - TMPD N,N,N,,N,-tetramethyl-p-phenylenediamine - Z secondary electron donor of PS II     

A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i−1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i–1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C chemical shifts with the carbonyl (¹³C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i–1CA_i often allows resolution of the ambiguity by linking the ¹³C and ¹³C spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns.     

Source of Nitrous Oxide Emissions during the Cow Manure Composting Process as Revealed by Isotopomer Analysis of and amoA Abundance in Betaproteobacterial Ammonia-Oxidizing Bacteria

A molecular analysis of betaproteobacterial ammonia oxidizers and a N₂O isotopomer analysis were conducted to study the sources of N₂O emissions during the cow manure composting process. Much NO₂⁻-N and NO₃⁻-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the ¹⁵N site preference within the asymmetric N₂O molecule (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N₂O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO₂⁻-N or NO₃⁻-N after turning was identified as the main source of N₂O emissions. The site preference and bulk δ¹⁵N results also indicate that the rate of N₂O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N₂O emissions between the turnings.The very sensitive greenhouse gas nitrous oxide (N₂O) has a 296 times higher impact than CO₂ (39) and is also responsible for ozone depletion (10). Agricultural activities such as the use of nitrate fertilizers, livestock production, and manure management, including composting, are known to be important sources of N₂O emissions (18). To devise a strategy to mitigate N₂O emissions, it is essential to understand its sources in detail. However, the sources of N₂O emissions during the composting process are still largely unclear.In the composting process, a part of NH₄⁺-N is known to be processed through nitrification-denitrification and emitted as N₂ and N₂O. Nitrous oxide is known to be generated through both the nitrification and denitrification processes as intermediate products or by-products. Nitrous oxide emission is a very complex process because denitrifying bacteria are phylogenetically diverse (60), and nitrifiers are also known to utilize the denitrification process even under aerobic conditions (42). It is thus very difficult to estimate the relative contributions of nitrification and denitrification in actual N₂O emissions from the environment. Until now, there has been insufficient knowledge about the relative contributions of these processes to N₂O emissions during the animal manure composting process. Measurement of the actual contributions of N₂O emissions from compost piles in the field is therefore critical to establishing a strategy of mitigating N₂O emissions.Recently, a high-precision analytical technique for determining intramolecular ¹⁵N site preference in asymmetric molecules of N₂O was developed (47). Since N₂O has two N atoms within the molecule (central and outer N), distribution of a stable isotope, ¹⁵N, results in the distribution of three isotopomers, such as ¹⁵N¹⁵NO, ¹⁵N¹⁴NO, and ¹⁴N¹⁵NO. By using this newly developed innovative technique, the latter two types of molecules, which exist abundantly in the environment, can be individually measured. The difference in δ¹⁵N between δ¹⁵N^α and δ¹⁵N^β is the so-called site preference (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively). The site preference enabled us to identify the source and sinks of N₂O in the environment (48, 49, 50, 56). Using this technique, Sutka et al. (44) found that the site preference for N₂O from hydroxylamine oxidation (∼33‰) and nitrite reduction (∼0‰) differs in a pure culture study and noted that this difference can be used to distinguish the relative contributions of nitrification and denitrification sources to N₂O emissions. There have still been only several reported studies which applied this measurement technique to field N₂O samples (48, 53) or referred to the relative contributions of nitrification and denitrification. To our knowledge, the present study is the first to apply this isotopomer analysis technique to the determination of N₂O sources in the composting process. We specifically used this technique to understand the actual contributions of nitrification and denitrification to N₂O emissions during the cow manure composting process.Ammonia oxidation, the conversion of ammonium to nitrite via hydroxylamine, is an initial step of the nitrification-denitrification process and is critical to the nitrogen cycle in the terrestrial environment (4, 24). In the nitrification process, N₂O is generated as a by-product when ammonia oxidizers convert hydroxylamine to nitrite (35). Since NO₂⁻-N and NO₃⁻-N accumulate in the latter stages of the composting process (29, 30), it is obvious that nitrifiers are active in compost piles. Therefore, it is important to clarify the role and significance of ammonia oxidizers in N₂O emissions during the composting process. However, since the pure culture isolation method is so difficult and time-consuming, little is known about these ammonia oxidizers. A molecular approach based on PCR has been recently developed and has to date been used to target the ammonia monooxygenase gene (amoA) or 16S rRNA gene of betaproteobacterial ammonia oxidizers in soil, wetlands, and marine sediments (2, 3, 6, 7, 13, 32, 52). Using these techniques, substantial information about uncultured ammonia-oxidizing bacteria (AOB) that are partially or wholly responsible for nitrification in the environment will become available. Since the microbial community drastically changes through the composting process (19, 29), and a high accumulation of nitrite or nitrate will occur, especially in the latter half of the process (30), we continuously sampled and analyzed the diversity and abundance of AOB throughout the process. Our objectives in this study were to elucidate the sources of N₂O emissions during the cow manure composting process by combining the isotopomer analysis and molecular analysis of betaproteobacterial AOB.     

Chronic nitrate additions dramatically increase the export of carbon and nitrogen from northern hardwood ecosystems

A long-term field experiment was initiated to simulate chronic atmospheric N deposition, a widespread phenomenon in industrial regions of the world. Eight years of experimental nitrate ( ) additions (3 g -N m^–2 per year) to four different northern hardwood forests located along a 500 km geographic gradient dramatically increased leaching losses of -N, dissolved organic carbon (DOC), and dissolved organic nitrogen (DON). During the last two water years, the average increase in solution -N and DON leaching from the -amended plots was 2.2 g N m^–2, equivalent to 72% of the annual experimental N addition. Results indicate that atmospheric N deposition may rapidly saturate some northern hardwood ecosystems across an entire biome in the upper Great Lakes Region of the USA. Changes in soil C and N cycling induced by chronic N deposition have the potential in this landscape to significantly alter the flux of DOC and DON from upland to aquatic ecosystems. Michigan Gradient study site characteristics are similar to those of European forests most susceptible to N saturation.     

The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

On the phytochrome phototransformation kinetics in mustard seedlings

Summary The in vivo phototransformation kinetics of mustard hook and cotyledon phytochrome exhibit a deviation from a single first order curve, quite similar to that for pumpkin hooks as reported in a previous paper (Boisard, Marmé and Schäfer, 1971). The P _frP_rkinetics can be characterized by the ratios _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II and where P _fr ^I and P _fr ^II are two populations of phytochrome molecules which convert to P _rwith a first order half-life of and . These ratios depend on the length of time of etiolation. The ratio _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II is independent of the amount of total P _frpresent at the beginning of the P _frP_rphototransformation after a non-saturating dose of red light. The half-lives of the two populations, however, depend on the concentration of total P _frinitially present. P _frP_rphototransformation kinetics with different light intensities show that reciprocity holds.