Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta.

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2-5, 12 and 13 cDNAs, produced the proteins in full (PGRP2-5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.

Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.

The combination of 13N-ammonia and 18F-FDG PET/CT in the identification of metabolic phenotype of primary human brain tumors.

AIM: This pilot study made a preliminary attempt to distinguish different metabolic phenotypes of primary human brain tumors with dual tracer 13N-ammonia and 18F-FDG. METHODS: 74 patients were included in this study including 12 benign meningiomas (B-MEN), 4 malignant meningiomas (M-MEN), 15 low-grade gliomas (LGG), 32 high-grade gliomas (HGG) and 11 primary central nervous system lymphomas (PCNSL). All patients underwent 13N-ammonia and 18F-FDG PET imaging. Semi-quantification analysis by tumor-to-gray matter (T/G) ratios was used for the evaluation of tracer uptakes. After the calculation of canonical discriminant functions, cross validation was done for all cases to evaluate the differential efficacy of dual tracers. RESULTS: According to the visual analysis, B-MEN were characterized by lower uptake of 18F-FDG and higher uptake of 13N-ammonia, while PCNSLs displayed contrary results. Both M-MEN and HGG had higher uptake of 18F-FDG and 13N-ammonia, while LGG displayed negative results for both tracers. According to the T/G ratios analysis, the accuracy of predicted tumor classification by means of canonical discriminant analysis for B-MEN, LGG, HGG and PCNSL was 91.7 %, 100 %, 84.4 % and 93.3 % respectively; the overall accuracy was 90.5 %. CONCLUSION: The combination of dual tracer 13N-ammonia and 18F-FDG has a certain potential in distinguishing different types of brain tumors (meningiomas, gliomas and PCNSL). However, an advantage of the additional use of 13N-ammonia PET compared to a combined diagnosis with MRI and 18F-FDG PET could not be demonstrated and requires further studies.

Quantifying N-loss by root abscission: consequences for wheat N budgets and δ15N values.

Lower plant δ15N values relative to source δ15N are commonly attributed to 15N efflux. We determined the extent to which root abscission contributes to plant N-loss and consequences for plant δ15N. Wheat (Triticum aestivum L. cv. SST015) was grown in hydroponics with direct aeration, aeration constrained within a pipe and circulation of nutrient solution through sand, representing three levels of stability for root growth. The δ15N of nutrient solutions and root fragments were periodically determined, as well as root and shoot δ15N. Plants in solution had significantly more negative δ15N (-8.9 and -9.2‰) than plants in sand (-6.9‰), suggesting greater 15N-loss; root fragments were major biomass- (six-fold greater than root dry weight) and N-loss (two-fold greater than plant net N uptake) pathways in solution. These plants had more ephemeral roots and two-fold more root tips than the sand treatment. We estimated that root fragment loss decreased plant δ15N by at least -3.7, -2.6 and -1.0‰ in the direct, pipe and sand treatments, respectively. Positive nutrient solution δ15N in all treatments relative to the source δ15N suggests that plant N, probably derived from efflux, was present in solution. Despite this, root abscission and root turnover are also important N-loss pathways in plants, while plant δ15N values are probably influenced by a combination of root abscission and N efflux.

Dynamics of amino acid redistribution in the carnivorous Venus flytrap (Dionaea muscipula) after digestion of 13 C/15 N-labelled prey.

Amino acids represent an important component in the diet of the Venus flytrap (Dionaea muscipula), and supply plants with much needed nitrogen resources upon capture of insect prey. Little is known about the significance of prey-derived carbon backbones of amino acids for the success of Dionaea's carnivorous life-style. The present study aimed at characterizing the metabolic fate of 15 N and 13 C in amino acids acquired from double-labeled insect powder. We tracked changes in plant amino acid pools and their δ13 C- and δ15 N-signatures over a period of five weeks after feeding, as affected by contrasting feeding intensity and tissue type (i.e., fed and non-fed traps and attached petioles of Dionaea). Isotope signatures (i.e., δ13 C and δ15 N) of plant amino acid pools were strongly correlated, explaining 60% of observed variation. Residual variation was related to contrasting effects of tissue type, feeding intensity and elapsed time since feeding. Synthesis of nitrogen-rich transport compounds (i.e., amides) during peak time of prey digestion increased 15 N- relative to 13 C- abundances in amino acid pools. After completion of prey digestion, 13 C in amino acid pools was progressively exchanged for newly fixed 12 C. The latter process was most evident for non-fed traps and attached petioles of plants that had received ample insect powder. We argue that prey-derived amino acids contribute to respiratory energy gain and loss of 13 CO2 during conversion into transport compounds (i.e., 2 days after feeding), and that amino-nitrogen helps boost photosynthetic carbon gain later on (i.e., 5 weeks after feeding).

Dry season limnological conditions and basin geology exhibit complex relationships with δ13C and δ15N of carbon sources in four Neotropical floodplains.

Studies in freshwater ecosystems are seeking to improve understanding of carbon flow in food webs and stable isotopes have been influential in this work. However, variation in isotopic values of basal production sources could either be an asset or a hindrance depending on study objectives. We assessed the potential for basin geology and local limnological conditions to predict stable carbon and nitrogen isotope values of six carbon sources at multiple locations in four Neotropical floodplain ecosystems (Paraná, Pantanal, Araguaia, and Amazon). Limnological conditions exhibited greater variation within than among systems. δ15N differed among basins for most carbon sources, but δ13C did not (though high within-basin variability for periphyton, phytoplankton and particulate organic carbon was observed). Although δ13C and δ15N values exhibited significant correlations with some limnological factors within and among basins, those relationships differed among carbon sources. Regression trees for both carbon and nitrogen isotopes for all sources depicted complex and in some cases nested relationships, and only very limited similarity was observed among trees for different carbon sources. Although limnological conditions predicted variation in isotope values of carbon sources, we suggest the resulting models were too complex to enable mathematical corrections of source isotope values among sites based on these parameters. The importance of local conditions in determining variation in source isotope values suggest that isotopes may be useful for examining habitat use, dispersal and patch dynamics within heterogeneous floodplain ecosystems, but spatial variability in isotope values needs to be explicitly considered when testing ecosystem models of carbon flow in these systems.

Residual Activity Correction in Quantitative Myocardial Perfusion 13N-Ammonia PET Imaging: A Study in Post-MI Patients.

BACKGROUND/INTRODUCTION/AIM: Positron emission tomography (PET) is the gold standard for the quantification of myocardial blood flow (MBF). A standard PET scan is acquired in two phases (rest and pharmacological stress). 13N-ammonia is a perfusion radiotracer that may show residual activity, which may affect MBF estimation during the second phase of the scan. An algorithm for residual activity correction (RAC) is available when reconstruction is performed using Syngo MBF (by Siemens). The aim of this study was to evaluate differences in MBF estimation with and without RAC by Syngo MBF in patients with a previous MI using 13N-ammonia PET. METHODS: MBF was evaluated by 13N-ammonia PET in a group of 25 patients with a history of MI. Dynamic MBF measurements were analyzed with Syngo Dynamic PET, with and without RAC, and the results were evaluated with statistical methods. RESULTS: Significant differences in stress phase MBF after RAC were identified in the left anterior descending coronary artery (LAD) territory (p=0.0425) and the right coronary artery (RCA) territory (p=0.004). A trend towards significance was identified in the global polar plot (p=0.049). No statistically significant difference was found in the left circumflex artery (LCx) territory (p=0.333). CONCLUSION: Syngo Dynamic PET, through its RAC function, can be a useful adjunct in assessing second-phase MBF of primarily the RCA territory and secondarily the global polar plot and LAD territory but not the LCx territory.

Organ Distribution of 13N Following Intravenous Injection of [13N]Ammonia into Portacaval-Shunted Rats.

Ammonia is neurotoxic, and chronic hyperammonemia is thought to be a major contributing factor to hepatic encephalopathy in patients with liver disease. Portacaval shunting of rats is used as an animal model to study the detrimental metabolic effects of elevated ammonia levels on body tissues, particularly brain and testes that are deleteriously targeted by high blood ammonia. In normal adult rats, the initial uptake of label (expressed as relative concentration) in these organs was relatively low following a bolus intravenous injection of [13N]ammonia compared with lungs, kidneys, liver, and some other organs. The objective of the present study was to determine the distribution of label following intravenous administration of [13N]ammonia among 14 organs in portacaval-shunted rats at 12 weeks after shunt construction. At an early time point (12 s) following administration of [13N]ammonia the relative concentration of label was highest in lung with lower, but still appreciable relative concentrations in kidney and heart. Clearance of 13N from blood and kidney tended to be slower in portacaval-shunted rats versus normal rats during the 2-10 min interval after the injection. At later times post injection, brain and testes tended to have higher-than-normal 13N levels, whereas many other tissues had similar levels in both groups. Thus, reduced removal of ammonia from circulating blood by the liver diverts more ammonia to extrahepatic tissues, including brain and testes, and alters the nitrogen homeostasis in these tissues. These results emphasize the importance of treatment paradigms designed to reduce blood ammonia levels in patients with liver disease.

Decreased ¹³N-labeled ammonia uptake in the ipsilateral and contralateral hemispheres following carotid endarterectomy.

Carotid artery plaques are a leading cause of ischemic stroke, and carotid endarterectomy (CEA) is one of the major treatment approaches for this disease. Changes in cerebral metabolism following CEA remain unclear. The present study aimed to evaluate the effect of cerebral ammonia metabolism following CEA using 13N­labeled ammonia positron emission tomography (PET) in humans. A total of 20 patients were enrolled in the present study, with a mean age of 59.5 years, comprising 16 males and four females. Of these patients, eight underwent right CEA and 12 underwent left CEA. The rate of carotid artery stenosis was between 50­69% in six of the patients, between 70­99% in 11 of the patients and was at 100% (thrombosis) in three of the patients, measured by computerised tomography digital subtraction angiography prior to CEA. 13N­labeled ammonia (137 MBq) PET scanning was performed prior and subsequent to CEA surgery for each patient. The first ammonia PET scan was performed 1 day prior to CEA, while the second PET scan was performed 1­4 weeks following CEA. Following injection of 13N­labeled ammonia, static PET was acquired for 10 min. The region of interest (ROI), covering the major cerebral hemisphere, was selected and ammonia uptake in the ROI was determined in the ipsilateral and contralateral hemispheres. No hyperperfusion syndrome was observed in the patients subsequent to CEA. No significant change in cerebral hemisphere ammonia uptake was observed between the ipsilateral and contralateral hemispheres prior to (ratio =0.98; P>0.01) or following (ratio =1.09; P>0.01) CEA. Ammonia uptake in the ipsilateral and contralateral hemispheres was significantly reduced to 23.2 and 23.5%, respectively, following CEA. Using 13N­labeled ammonia PET to evaluate cerebral ammonia metabolism following CEA in patients with severe carotid artery stenosis, the present study demonstrated that uptake of ammonia in the ipsilateral and contralateral hemispheres was significantly reduced.

Assessing nitrification and denitrification in a paddy soil with different water dynamics and applied liquid cattle waste using the ¹5N isotopic technique.

Using livestock wastewater for rice production in paddy fields can remove nitrogen and supplement the use of chemical fertilizers. However, paddy fields have complicated water dynamics owing to varying characteristics and would influence nitrogen removal through nitrification followed by denitrification. Quantification of nitrification and denitrification is of great importance in assessing the influence of water dynamics on nitrogen removal in paddy fields. In this study, nitrification and nitrate reduction rates with different water dynamics after liquid cattle waste application were evaluated, and the in situ denitrification rate was determined directly using the (15)N isotopic technique in a laboratory experiment. A significant linear regression correlation between nitrification and the nitrate reduction rate was observed and showed different regression coefficients under different water dynamics. The regression coefficient in the continuously flooded paddy soil was higher than in the drained-reflooded paddy soil, suggesting that nitrate would be consumed faster in the flooded paddy soil. However, nitrification was limited and the maximum rate was only 13.3 µg Ng(-1)day(-1) in the flooded paddy soil with rice plants, which limited the supply of nitrate. In contrast, the drained-reflooded paddy soil had an enhanced nitrification rate up to 56.8 µg Ng(-1)day(-1), which was four times higher than the flooded paddy soil and further stimulated nitrate reduction rates. Correspondingly, the in situ denitrification rates determined directly in the drained-reflooded paddy soil ranged from 5 to 1035 mg Nm(-2)day(-1), which was higher than the continuously flooded paddy soil (from 5 to 318 mg Nm(-2)day(-1)) during the vegetation period. The nitrogen removal through denitrification accounted for 38.9% and 9.9% of applied nitrogen in the drained-reflooded paddy soil and continuously flooded paddy soil, respectively.

Usefulness of (13)N-NH (3) PET in the evaluation of brain lesions that are hypometabolic on (18)F-FDG PET.

We investigated the usefulness of (13)N-NH(3) PET in characterizing brain lesions which show hypometabolism on (18)F-FDG PET. (13)N-NH(3) PET was performed in 18 patients with brain lesions (in 14 for initial diagnosis and in 4 for detection of astrocytoma recurrence) that showed hypometabolism compared with normal brain tissue on (18)F-FDG PET. The diagnoses were ten gliomas, one metastatic tumor, one dysembryoplastic neuroepithelial tumor (DNT), and six non-neoplastic lesions (including three cases of radiation necrosis, two cases of encephalitic foci, and one ischemic lesion). Diagnosis was verified by histopathological examination in 13 patients or was established by clinical follow-up and additional investigations in the remainder. Seven of 12 brain tumors (58%, sensitivity) showed increased (13)N-NH(3) uptake despite hypometabolism on (18)F-FDG PET. The three low-grade gliomas, one metastatic tumor, and one DNT showed decreased (13)N-NH(3) uptake. The mean (±SD) uptake ratio of (13)N-NH(3) was significantly higher than that of (18)F-FDG (1.24 ± 0.57 vs. 0.67 ± 0.21, P < 0.01) in the tumors. By contrast, all six non-neoplastic lesions showed decreased (13)N-NH(3) uptake (100% specificity). The mean (±SD) uptake ratio of (18)F-FDG and (13)N-NH(3) in the non-neoplastic lesions was 0.68 ± 0.15 and 0.70 ± 0.19, respectively, and there was no significant difference between them (P > 0.05). The mean (±SD) uptake ratio of (13)N-NH(3) in the tumors was significantly higher than that in the non-neoplastic lesions (1.24 ± 0.53 vs. 0.70 ± 0.19, P < 0.05). The preliminary results of this study suggest that (13)N-NH(3) PET may be helpful to detect and differentiate brain tumors which show hypometabolism on (18)F-FDG PET from non-neoplastic lesions with high specificity, especially for cerebral astrocytomas, but the sensitivity is relatively limited.

Imprint of denitrifying bacteria on the global terrestrial biosphere.

Loss of nitrogen (N) from land limits the uptake and storage of atmospheric CO(2) by the biosphere, influencing Earth's climate system and myriads of the global ecological functions and services on which humans rely. Nitrogen can be lost in both dissolved and gaseous phases; however, the partitioning of these vectors remains controversial. Particularly uncertain is whether the bacterial conversion of plant available N to gaseous forms (denitrification) plays a major role in structuring global N supplies in the nonagrarian centers of Earth. Here, we use the isotope composition of N ((15)N/(14)N) to constrain the transfer of this nutrient from the land to the water and atmosphere. We report that the integrated (15)N/(14)N of the natural terrestrial biosphere is elevated with respect to that of atmospheric N inputs. This cannot be explained by preferential loss of (14)N to waterways; rather, it reflects a history of low (15)N/(14)N gaseous N emissions to the atmosphere owing to denitrifying bacteria in the soil. Parameterizing a simple model with global N isotope data, we estimate that soil denitrification (including N(2)) accounts for approximately 1/3 of the total N lost from the unmanaged terrestrial biosphere. Applying this fraction to estimates of N inputs, N(2)O and NO(x) fluxes, we calculate that approximately 28 Tg of N are lost annually via N(2) efflux from the natural soil. These results place isotopic constraints on the widely held belief that denitrifying bacteria account for a significant fraction of the missing N in the global N cycle.

In vivo monitoring of extracellular 13N-glutamine derived from blood-borne 13N-ammonia in rat striatum using microdialysis with radio-LC method.

Glutamine synthetase (GS) is selectively localized in astrocytes and has important roles in the central nervous system (CNS). Cerebral extracellular excess ammonia and glutamate are taken up by astrocytes and converted to glutamine via GS to protect the CNS against neurotoxicity. In this study, we monitored cerebral extracellular 13N-glutamine derived from 13N-ammonia as a potential marker for astroglial metabolism using in vivo microdialysis combined with ultra performance liquid chromatography-radiometric detection. This method allowed rapid and highly sensitive radiometric analysis of 13N-ammonia and its metabolite, 13N-glutamine, in striatal extracellular fluid with good time resolution. Inhibition of GS with methionine sulfoximine resulted in a decrease of extracellular 13N-glutamine accompanied by an increase of 13N-ammonia as compared with control. Fluorocitrate, a selective inhibitor of glial metabolism, also decreased 13N-glutamine production and increased unmetabolized 13N-ammonia. In contrast, 13N-glutamine was increased with 5 mmol/kg of ammonium acetate without significant changes in 13N-ammonia as compared with control. These results suggest that the concentration of extracellular 13N-glutamine strongly reflects the biological changes in the metabolic activity of astroglial cells.

Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography.

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. Unlike magnetic resonance imaging (MRI) or computerized tomography (CT), which mainly provide detailed anatomical images, PET can measure chemical changes that occur before macroscopic anatomical signs of a disease are observed. PET is emerging as a revolutionary method for measuring body function and tailoring disease treatment in living subjects. The development of synthetic strategies for the synthesis of new positron-emitting molecules is, however, not trivial. This Review highlights key aspects of the synthesis of PET radiotracers with the short-lived positron-emitting radionuclides (11)C, (18)F, (15)O, and (13)N, with emphasis on the most recent strategies.

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport.

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry.

We have established a protocol to study the kinetics of incorporation of 15N into glutamine (Gln), glutamic acid (Glu), alanine (Ala) and proline (Pro) in Aedes aegypti females. Mosquitoes were fed 3% sucrose solutions containing either 80 mM 15NH4Cl or 80 mM glutamine labeled with 15N in either the amide nitrogen or in both amide and amine nitrogens. In some experiments, specific inhibitors of glutamine synthetase or glutamate synthase were added to the feeding solutions. At different times post feeding, which varied between 0 and 96 h, the mosquitoes were immersed in liquid nitrogen and then processed. These samples plus deuterium labeled internal standards were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. The quantification of 15N-labeled and unlabeled amino acids was performed by using mass spectrometry techniques. The results indicated that the rate of incorporation of 15N into amino acids was rapid and that the label first appeared in the amide side chain of Gln and then in the amino group of Gln, Glu, Ala and Pro. The addition of inhibitors of key enzymes related to the ammonia metabolism confirmed that mosquitoes efficiently metabolize ammonia through a metabolic route that mainly involves glutamine synthetase (GS) and glutamate synthase (GltS). Moreover, a complete deduced amino acid sequence for GltS of Ae. aegypti was determined. The sequence analysis revealed that mosquito glutamate synthase belongs to the category of NADH-dependent GltS.

Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase.

A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies.

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.

Supplementing barley or rapeseed meal to dairy cows fed grass-red clover silage: II. Amino acid profile of microbial fractions.

Four ruminally cannulated dairy cows were used to examine the effect of diet on the AA composition of rumen bacteria and protozoa, and the flow of microbial and nonmicrobial AA entering the omasal canal. Cows were offered grass-red clover silage alone, or that supplemented with 5.1 kg DM of barley, 1.9 kg DM of rapeseed meal, or 5.1 kg DM of barley and 1.9 kg DM of rapeseed meal according to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. During the first 10 d of each period, cows had free access to silage and, thereafter intake was restricted to 95% of ad libitum intake. Postruminal digesta flow was assessed using the omasal canal sampling technique in combination with a triple marker method. Liquid- (LAB) and particle- (PAB) associated bacteria were isolated from digesta in the reticulorumen and protozoa from digesta entering the omasal canal. Microbial protein flow was determined using 15N as a microbial marker. Flows of AA entering the omasal canal were similar in cows fed silage diets supplemented with barley or rapeseed meal. However, rapeseed meal increased nonmicrobial AA flow while barley increased the flow of AA associated with LAB and protozoa. Diet had negligible effects on the AA profile of microbial fractions. Comparison of AA profiles across diets indicated differences between LAB and PAB for 10 out of 17 AA measured. Rumen bacteria and protozoa were found to be different for 14 out of 15 AA measured. For grass silage-based diets, energy and protein supplementations appear to alter postruminal AA supply through modifications in the proportionate contribution of microbial and nonmicrobial pools to total protein flow rather than as a direct result of changes in the AA profile of microbial protein.

Short-term changes in xylem N compounds in Lolium perenne following defoliation.

Previous studies have indicated that an increased asparagine to glutamine ratio (Asn : Gln) occurs in the xylem fluid of Lolium perenne 24 h after defoliation. However, the absolute changes in Asn and Gln leading to the increased Asn : Gln ratio are unknown. The present study tested the hypotheses that: (1) defoliation-induced changes in xylem amino acid composition occur in L perenne within the first 24 h following defoliation, irrespective of phasing with respect to the diurnal light/dark cycle; and (2) the increase in Asn : Gln ratio in the xylem fluid of L perenne following defoliation is due to an increase in Asn content. Plants of L perenne L. 'Aurora' were grown in flowing solution culture for 40 d. Plants were then either left intact, defoliated at the end of the light period or defoliated at the end of the dark period. 15N-labelled NO3- was supplied following defoliation to discriminate between the recovery of N absorbed prior to, and following, defoliation. Xylem samples were collected over the subsequent 24 h period with amino acids speciated by GC-MS. There was support for the first hypothesis: increased Asn : Gln ratios occurred within the first 24 h, irrespective of the phasing of defoliation with respect to light/dark cycles. The second hypothesis was not supported: the concentration of all amino acids in the xylem exudate declined after defoliation, and the increased Asn : Gln ratio was accounted for by a disproportionately large reduction in Gln levels. Low concentrations of amino acids in the xylem of defoliated plants precluded accurate discrimination of their nitrogen content into pre- and post-defoliation sources.