Effects of light spectra and 15N pulses on growth, leaf morphology, physiology, and internal nitrogen cycling in Quercus variabilis Blume seedlings.

Light spectra of sunlight transmittance can generate an interactive effect with deposited nitrogen (N) on regenerated plants across varied shading conditions. Total N content in understory plants can be accounted for by both exogeneous and endogenous sources of derived N, but knowledge about the response of inner N cycling to interactive light and N input effects is unclear. We conducted a bioassay on Chinese cork oak (Quercus variabilis Blume) seedlings subjected to five-month N pulsing with 15NH4Cl (10.39 atom %) at 120 mg 15N plant-1 under the blue (48.5% blue, 33.7% green, and 17.8% red), red (14.6% blue, 71.7% red, 13.7% green), and green (17.4% blue, 26.2% red, 56.4% green) lighting-spectra. Half of the seedlings were fed twice a week using a 250 ppm N solution with micro-nutrients, while the other half just received distilled water. Two factors showed no interaction and neither affected growth and morphology. Compared to the red-light spectrum, that in blue light increased chlorophyll and soluble protein contents and glutamine synthetase (GS) activity, root N concentration, and N derived from the pulses. The green-light spectrum induced more biomass allocation to roots and a higher percentage of N derived from internal reserves compared to the red-light spectrum. The 15N pulses reduced the reliance on N remobilization from acorns but strengthened shoot biomass, chlorophyll content, GS activity, and N concentration. In conclusion, light spectrum imposed an independent force from external N pulse to modify the proportion of N derived from internal sources in total N content in juvenile Q. variabilis.

15N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker.

The dinoflagellate Alexandrium minutum (A. minutum) which can produce paralyticshellfish toxins (PSTs) is often used as a model to study the migration, biotransformation,accumulation, and removal of PSTs. However, the mechanism is still unclear. To provide a new toolfor related studies, we tried to label PSTs metabolically with 15N stable isotope to obtain 15N-PSTsinstead of original 14N, which could be treated as biomarker on PSTs metabolism. We then culturedthe A. minutum AGY-H46 which produces toxins GTX1-4 in f/2 medium of different 15N/Pconcentrations. The 15N-PSTs' toxicity and toxin profile were detected. Meanwhile, the 15N labelingabundance and 15N atom number of 15N-PSTs were identified. The 14N of PSTs produced by A.minutum can be successfully replaced by 15N, and the f/2 medium of standard 15N/P concentrationwas the best choice in terms of the species' growth, PST profile, 15N labeling result and experimentcost. After many (>15) generations, the 15N abundance in PSTs extract reached 82.36%, and the 15Natom number introduced into GTX1-4 might be 4⁻6. This paper innovatively provided the initialevidence that 15N isotope application of labeling PSTs in A. minutum is feasible. The 15N-PSTs asbiomarker can be applied and provide further information on PSTs metabolism.

Selective permeability of mouse blood-aqueous barrier as determined by 15N-heavy isotope tracing and mass spectrometry.

The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier's selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (15N) atoms. Following systemic injection of this "heavy" serum in mice, the 15N-to-endogenous nitrogen-14 (14N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the 15N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus).

The diet-tissue discrimination factor is the amount by which a consumer's tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.

[The evaluation of snail host-trematode parasite trophic relationships using stable isotope analysis].

Stable isotope ratios of carbon (13C/12C, δ13C) and nitrogen (15N/14N, δ15N) in snail-host tissue (the foot and hepatopancreas) and trematode parasites on two stages of their life cycle were analyzed. Trophic structure in co-occurring trematode larvae was examined in the following species: five species of cercariae (Echinoparyphium recurvatum, Hypodereum conoideum, Plagiorchis mutationis, Diplostomum chromatophorum and D. volvens) and two species of metacercariae (Cotylurus cornutus and Echinoparyphium recurvatum) within two closely related snail hosts Lymnaea stagnalis and L. tumida using stable isotope analyses. Snail and parasite sampling was conducted in a riverine portion of the Kargat River of the Lake Chany basin, in the south of Western Siberia (54 degrees 37'76"N, 78 degrees 13'07" E), in August 2009. Four out of five studied cercariae species were depleted in 15N as well as in 13C relatively to snail hosts tissues (foot and hepatopancreas), supporting our previously published data for Plagiorchis mutationis and Echinoparyphium recurvatum cercariae. Such fractionation of cercariae is untypical of the commonly observed relationship between consumers and their food. D. chromatophorum cercariae had demonstrated an insufficient enrichment in δ15N volume in relation to the hepatopancreas. Both two species of metacercariae (C. cornutus and E. recurvatum) showed a significant enrichment in δ15N volume relatively to the host tissue consumed (fractionation values ranging from 1.5 to 4 per thousand depending on the species). The differences in δ15N and δ13C volume between cercariae and metacercariae observed in this study illustrate the complexity of the host--parasite trophic relationships. Such isotopic differences between cercariae and metacercariae can probably be explained by selective consumption of specific amino acids or lipids or by changes in metabolism associated with the life cycle of the parasite. The present study represents the first comparative analysis of trophic relationships between the host and the endoparasite at different life cycle stages. It demonstrates the potency of the stable isotope analyses for understanding trophic relationships in multispecies parasite communities.

Combined application of targeted and untargeted proteomics identifies distinct metabolic alterations in the tetraacetylphytosphingosine (TAPS) producing yeast Wickerhamomyces ciferrii.

The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production. BIOLOGICAL SIGNIFICANCE: First quantitative proteome study for a phytosphingosine-producing yeast.

Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized (1)H and (15)N NMR spectroscopy.

Brain glutamine synthetase (GS) is an integral part of the glutamate-glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. (15)N MRS is an alternative approach to (13)C MRS. Incorporation of labeled (15)N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate-glutamine cycle. To measure (15)N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel (15)N pulse sequence to simultaneously detect, for the first time, [5-(15)N]Gln and [2-(15)N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized (1)H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of (1)H and (15)N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 µmol/g per minute), apparent neurotransmission (0.26±0.030 µmol/g per minute), glutamate dehydrogenase (0.029±0.002 µmol/g per minute), and net glutamine accumulation (0.033±0.001 µmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification.

Effect of ezetimibe-simvastatine over endothelial dysfunction in dyslipidemic patients: assessment by 13N-ammonia positron emission tomography.

BACKGROUND: Dyslipidemias constitute an independent risk factor for the development of atherogenesis and they also predispose to the development of endothelial dysfunction (ED). Using PET with (13)N-ammonia, it is possible to quantify myocardial blood flow (MBF) in mL/min/g and to quantitatively evaluate ED. With the use of lipid lowering therapy it is possible to reduce ED and increase the MBF and the endothelial-dependent vasodilation index (ENDEVI). In this study, we aimed to evaluate with (13)N-ammonia PET the benefic effects of the combined treatment ezetimibe/simvastatine on the endothelial function of dyslipidemic patients after 8 weeks of treatment. MATERIAL AND METHODS: Fourteen consecutive patients with dyslipidemia diagnosis and 17 healthy volunteers were studied with a three phase [rest, Cold Pressor Test (CPT), and adenosine-induced hyperemia] (13)N-ammonia PET for MBF quantification assessment. A second PET study was performed in the dyslipidemic group after 8 weeks of treatment with ezetimibe/simvastatine (10/40 mg). Myocardial flow reserve (MFR), ENDEVI, and %ΔMBF were calculated. RESULTS: Total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides concentrations were markedly altered in the dyslipidemic group and after 8 weeks of treatment these values improved. Dyslipidemic patients showed endothelial dysfunction when compared with the control group, (MFR 2.79 ± 0.94 vs 3.15 ± 0.48, P < 0.05 ; ENDEVI 1.28 ± 0.25 vs 1.53 ± 0.24, P < 0.05; and %ΔMBF 29.08 ± 24.62 vs 53 ± 24.60%, P < 0.05, respectively). After 8 weeks of treatment, we found a significant increase in all the endothelial function markers (MFR: 3.14 ± 0.86, P < 0.05, ENDEVI 1.65 ± 0.23, P < 0.05; %ΔMBF: 65.21 ± 23.43, P < 0.05). CONCLUSIONS: Dyslipidemic patients show endothelial dysfunction measured with (13)N-ammonia PET. Treatment with ezetimibe/simvastatine was effective improving the lipid profile as well as the endothelial function of these patients. PET may be a useful tool to monitor vascular reactivity and regression/progression of coronary atherosclerosis after pharmacologic interventions.

Glutamine: precursor or nitrogen donor for citrulline synthesis?

Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

Nitrogen removal during summer and winter in a primary facultative waste stabilization pond: preliminary findings from (15)N-labelled ammonium tracking techniques.

Nitrogen removal mechanisms and pathways within WSP have been the focus of much research over the last 30 years. Debates and theories postulated continue to refine our knowledge regarding the cycling and removal pathways for this important nutrient, but a succinct answer has yet to be provided for holistic nitrogen removal. In this study, two experimental runs using labelled (15)N as a stable isotope tracking technique were conducted on a pilot-scale primary facultative WSP in the UK; one in the summer of 2006, and the other in the winter of 2007. An ammonium chloride ((15)NH(4)Cl) spike was prepared as the slug for each experimental run, which also contained rhodamine WT to act as a dye tracer enabling the hydraulic characteristics of the pond to be mapped. Initial results from the study are reported here, and findings are compared and contrasted. Preliminary findings reveal that a greater proportion of (15)N is incorporated into the algal biomass by assimilation and subsequent release as soluble organic nitrogen in summer than in winter. (15)N ammonium passes out of the system much sooner and in a much higher proportion in the winter than in summer.

The determination of biurea: a novel method to discriminate between nitrofurazone and azodicarbonamide use in food products.

Recently doubts have arisen on the usefulness of semicarbazide as marker residue for the illegal use of the antibiotic nitrofurazone (NFZ) in aquaculture and poultry production. Most notably azodicarbonamide (ADC) has been implicated as an alternative source of semicarbazide. ADC is used in some countries as a dough conditioner at concentrations up to 45 mg kg(-1). The use of ADC-treated flour or dough in coated or breaded food products may generate false non-compliant results in the analytical method for nitrofurazone metabolites, which is currently in use. During the dough preparation process ADC is largely reduced to biurea, which can be considered as an appropriate marker residue of ADC. Thus far no methods have been published for the determination of biurea in food commodities. Due to its polar nature it is very difficult to generate sufficient retention on conventional C18 HPLC columns. With a TSK amide HILIC type column good retention was obtained. A straightforward extraction-dilution protocol was developed. Using a mixture of dimethyl formamide and water biurea was nearly quantitatively extracted from a variety of fresh, coated and processed products. Mass spectrometric detection was performed with positive electrospray ionisation. The sensitivity and selectivity of the mass spectrometer for biurea was very good, allowing detection at concentrations as low as 10 microg kg(-1). However, in some extracts severe ion suppression effects was observed. To overcome the implications of ion suppression on the quantitative performance of the method an isotopically-labelled biurea internal standard was synthesized and incorporated in the method. The method developed can be used effectively in nitrofurazone analysis to eliminate the risk of false non-compliant results due to the presence of azodicarbonamide-treated components in the food product.