Foliar and soil δ15N values reveal increased nitrogen partitioning among species in diverse grassland communities

Plant and soil nitrogen isotope ratios (δ¹⁵N) were studied in experimental grassland plots of varying species richness. We hypothesized that partitioning of different sources of soil nitrogen among four plant functional groups (legumes, grasses, small herbs, tall herbs) should increase with diversity. Four years after sowing, all soils were depleted in ¹⁵N in the top 5 cm whereas in non‐legume plots soils were enriched in ¹⁵N at 5–25 cm depth. Decreasing foliar δ¹⁵N and Δδ¹⁵N (= foliar δ¹⁵N ? soil δ¹⁵N) values in legumes indicated increasing symbiotic N₂ fixation with increasing diversity. In grasses, foliar Δδ¹⁵N also decreased with increasing diversity suggesting enhanced uptake of N depleted in ¹⁵N. Foliar Δδ¹⁵N values of small and tall herbs were unaffected by diversity. Foliar Δδ¹⁵N values of grasses were also reduced in plots containing legumes, indicating direct use of legume‐derived N depleted in ¹⁵N. Increased foliar N concentrations of tall and small herbs in plots containing legumes without reduced foliar δ¹⁵N indicated that these species obtained additional mineral soil N that was not consumed by legumes. These functional group and species specific shifts in the uptake of different N sources with increasing diversity indicate complementary resource use in diverse communities.     

Variation of bone collagen amino acid δ13C values in archaeological humans and fauna with different dietary regimes: developing frameworks of dietary discrimination

We present bone collagen amino acid (AA) δ(13)C values for a range of archaeological samples representing four "benchmark" human diet groups (high marine protein consumers, high freshwater protein consumers, terrestrial C(3) consumers, and terrestrial C(4) consumers), a human population with an "unknown" diet, and ruminants. The aim is to establish an interpretive palaeodietary framework for bone collagen AA δ(13)C values, and to assess the extent to which AA δ(13)C values can provide additional dietary information to bulk collagen stable isotope analysis. Results are analyzed to determine the ability of those AAs for which we have a complete set, to discriminate between the diet groups. We show that very strong statistical discrimination is obtained for all interdiet group comparisons. This is often obvious from suitably chosen bivariate plots using δ(113)C values that have been normalized to compensate for interdiet group differences in bulk δ(13)C values. Bi-plots of non-normalized phenylalanine and valine δ(13)C values are useful for distinguishing aquatic diets (marine and freshwater) from terrestrial diets. Our interpretive framework uses multivariate statistics (e.g., discriminant analysis) to optimize the separation of the AA δ(13)C values of the "benchmark"' diet groups, and is capable of accurately assigning external samples to their expected diet groups. With a growing body of AA δ(13)C values, this method is likely to enhance palaeodietary research by allowing the "unknown" diets of populations under investigation to be statistically defined relative to the well-characterized or "known" diets of previously investigated populations.     

δ15N values of tropical savanna and monsoon forest species reflect root specialisations and soil nitrogen status

A large number of herbaceous and woody plants from tropical woodland, savanna, and monsoon forest were analysed to determine the impact of environmental factors (nutrient and water availability, fire) and biological factors (microbial associations, systematics) on plant delta(15)N values. Foliar delta(15)N values of herbaceous and woody species were not related to growth form or phenology, but a strong relationship existed between mycorrhizal status and plant delta(15)N. In woodland and savanna, woody species with ectomycorrhizal (ECM) associations and putative N(2)-fixing species with ECM/arbuscular (AM) associations had lowest foliar delta(15)N values (1.0-0.6 per thousand ), AM species had mostly intermediate delta(15)N values (average +0.6 per thousand ), while non-mycorrhizal Proteaceae had highest delta(15)N values (+2.9 to +4.1 per thousand ). Similar differences in foliar delta(15)N were observed between AM (average 0.1 and 0.2 per thousand ) and non-mycorrhizal (average +0.8 and +0.3 per thousand ) herbaceous species in woodland and savanna. Leguminous savanna species had significantly higher leaf N contents (1.8-2.5% N) than non-fixing species (0.9-1.2% N) indicating substantial N acquisition via N(2) fixation. Monsoon forest species had similar leaf N contents (average 2.4% N) and positive delta(15)N values (+0.9 to +2.4 per thousand ). Soil nitrification and plant NO(3)(-) use was substantially higher in monsoon forest than in woodland or savanna. In the studied communities, higher soil N content and nitrification rates were associated with more positive soil delta(15)N and plant delta(15)N. In support of this notion, Ficus, a high NO(3)(-) using taxa associated with NO(3)(-) rich sites in the savanna, had the highest delta(15)N values of all AM species in the savanna. delta(15)N of xylem sap was examined as a tool for studying plant delta(15)N relations. delta(15)N of xylem sap varied seasonally and between differently aged Acacia and other savanna species. Plants from annually burnt savanna had significantly higher delta(15)N values compared to plants from less frequently burnt savanna, suggesting that foliar (15)N natural abundance could be used as marker for assessing historic fire regimes. Australian woodland and savanna species had low leaf delta(15)N and N content compared to species from equivalent African communities indicating that Australian biota are the more N depauperate. The largest differences in leaf delta(15)N occurred between the dominant ECM Australian and African savanna (miombo) species, which were depleted and enriched in (15)N, respectively. While the depleted delta(15)N of Australian ECM species are similar to those of previous reports on ECM species in natural plant communities, the (15)N-enriched delta(15)N of African ECM species represent an anomaly.     

Effect of organic and inorganic fertigation on yields, δ15N values, and δ13C values of tomato (Lycopersicon esculentum Mill. cv. Saturn)

We examined the effects of fertilizer application, especially the effects of fertigation and types of fertilizer (inorganic and organic) on yields and ¹⁵N and ¹³C values of tomato (Lycopersicon esculentum Mill. cv. Saturn). Fertigation is a method in which an appropriate diluted liquid fertilizer is applied to the plants each time they are drip-irrigated. We developed a method of organic fertigation using corn steep liquor (CSL) as the liquid fertilizer, because it is an industrial byproduct of cornstarch manufacture and can be used very effectively. We compared fruit yield, mineral content, ¹⁵N value, and ¹³C value of tomatoes grown under three different fertilizer treatments, basal dressing: basal dressing with granular chemical fertilizer; inorganic fertigation: fertigation with liquid chemical fertilizer; and organic fertigation: fertigaion with CSL. Mineral contents of tomatoes grown with basal dressing were generally lower than those grown under either fertigation treatment. These results indicated that yields and mineral contents were influenced more by the method of fertilizer application than by whether the fertilizers were inorganic or organic. There were, however, significant differences in the ¹⁵N values of tomato fruits grown under different types of fertilizer applications, especially between inorganic and organic fertilizers. The ¹⁵N value of the chemical fertilizer used for basal dressing was 0.81 ± 0.45{}, that of the chemical fertilizer for fertigation was 0.00 ± 0.04{}, and that of CSL was 8.50 ± 0.71{}. The ¹⁵N values of the soils reflected the ¹⁵N values of the fertilizers. Moreover, the ¹⁵N values of the fruits corresponded to the ¹⁵N values of the applied fertilizers. The ¹⁵N values were 3.18 ± 1.34{} in the fruits grown with a basal dressing of chemical fertilizer, 0.30 ± 0.61 in those grown under inorganic fertigation, and 7.09 ± 0.68 in those grown under organic fertigation. On the other hand, although the ¹³C values in the soil also reflected the ¹³C values of the applied fertilizers, there was no significant difference in the ¹³C values of fruits among the different treatments. In conclusion, because the ¹⁵N values of fertilizers correlated well with those of the fruits, it may be possible to use ¹⁵N values as an indicator of organic products.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

How meaningful are Bayesian support values?

In this study, we used an empirical example based on 100 mitochondrial genomes from higher teleost fishes to compare the accuracy of parsimony-based jackknife values with Bayesian support values. Phylogenetic analyses of 366 partitions, using differential taxon and character sampling from the entire data matrix of 100 taxa and 7,990 characters, were performed for both phylogenetic methods. The tree topology and branch-support values from each partition were compared with the tree inferred from all taxa and characters. Using this approach, we quantified the accuracy of the branch-support values assigned by the jackknife and Bayesian methods, with respect to each of 15 basal clades. In comparing the jackknife and Bayesian methods, we found that (1) both measures of support differ significantly from an ideal support index; (2) the jackknife underestimated support values; (3) the Bayesian method consistently overestimated support; (4) the magnitude by which Bayesian values overestimate support exceeds the magnitude by which the jackknife underestimates support; and (5) both methods performed poorly when taxon sampling was increased and character sampling was not increases. These results indicate that (1) the higher Bayesian support values are inappropriate (in magnitude), and (2) Bayesian support values should not be interpreted as probabilities that clades are correctly resolved. We advocate the continued use of the relatively conservative bootstrap and jackknife approaches to estimating branch support rather than the more extreme overestimates provided by the Markov Chain Monte Carlo-based Bayesian methods.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned se- quences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitu- tion matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9.     

Stereospecific synthesis of α-methylated amino acids

Summary. Both 2,5-trans and 2,5-cis disubstituted 2-tert-butyl-5-(indol-3-yl)methylimidazolidin-4-ones were synthesised and their enolates were prepared using LDA. While the enolate of the 2,5-trans disubstituted derivative could not be methylated, the enolate of the cis-2,5-disubstituted derivative was successfully methylated with methyl iodide to a product which on hydrolysis gave enantiomerically pure α-methyl-L-tryptophan. Received October 31, 1998, Accepted July 23, 1999     

Variation in hair δ13C and δ15N values in long-tailed macaques (Macaca fascicularis) from Singapore

Much of the primatology literature on stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) has focused on African and New World species, with comparatively little research published on Asian primates. Here we present hair δ¹³C and δ¹⁵N isotope values for a sample of 33 long-tailed macaques from Singapore. We evaluate the suggestion by a previous researcher that forest degradation and biodiversity loss in Singapore have led to a decline in macaque trophic level. The results of our analysis indicated significant spatial variability in δ¹³C but not δ¹⁵N. The range of variation in δ¹³C was consistent with a diet based on C₃ resources, with one group exhibiting low values consistent with a closed canopy environment. Relative to other macaque species from Europe and Asia, the macaques from Singapore exhibited a low mean δ¹³C value but mid-range mean δ¹⁵N value. Previous research suggesting a decline in macaque trophic level is not supported by the results of our study.     

Stable isotope tracking of endangered sea turtles: validation with satellite telemetry and δ15N analysis of amino acids

Effective conservation strategies for highly migratory species must incorporate information about long-distance movements and locations of high-use foraging areas. However, the inherent challenges of directly monitoring these factors call for creative research approaches and innovative application of existing tools. Highly migratory marine species, such as marine turtles, regularly travel hundreds or thousands of kilometers between breeding and feeding areas, but identification of migratory routes and habitat use patterns remains elusive. Here we use satellite telemetry in combination with compound-specific isotope analysis of amino acids to confirm that insights from bulk tissue stable isotope analysis can reveal divergent migratory strategies and within-population segregation of foraging groups of critically endangered leatherback sea turtles (Dermochelys coriacea) across the Pacific Ocean. Among the 78 turtles studied, we found a distinct dichotomy in δ(15)N values of bulk skin, with distinct "low δ(15)N" and "high δ(15)N" groups. δ(15)N analysis of amino acids confirmed that this disparity resulted from isotopic differences at the base of the food chain and not from differences in trophic position between the two groups. Satellite tracking of 13 individuals indicated that their bulk skin δ(15)N value was linked to the particular foraging region of each turtle. These findings confirm that prevailing marine isoscapes of foraging areas can be reflected in the isotopic compositions of marine turtle body tissues sampled at nesting beaches. We use a Bayesian mixture model to show that between 82 and 100% of the 78 skin-sampled turtles could be assigned with confidence to either the eastern Pacific or western Pacific, with 33 to 66% of all turtles foraging in the eastern Pacific. Our forensic approach validates the use of stable isotopes to depict leatherback turtle movements over broad spatial ranges and is timely for establishing wise conservation efforts in light of this species' imminent risk of extinction in the Pacific.     

Stimulated uptake of amino acids by HeLa cells following ‘preloading’ in high concentrations of amino acids

HeLa cells exposed to 10mM leucine in medium for 30 mins at 37 °C showed greatly increased uptake of tritiated leucine when returned to medium containing 0.1mM levels of labelled amino acid. Further analysis of the phenomenon demonstrates that the steepness of the gradient is essential for rapid uptake to be observed, that no stereo-specificity is involved, and that the peak of uptake is followed by discharge with first order kinetics back to equilibrium. The effect of 14 different amino acids examined individually as 10mM supplements to the labelling medium ranged from slight reduction (lysine, proline) to complete suppression of the ‘stimulated uptake’ (phenylalanine, isoleucine).     

δ15N values of macroalgae as an indicator of the potential presence of waste disposal from land-based marine fish farms

The nitrogen isotope ratio (δ¹⁵N) in tissues of native macroalgae was evaluated as a means of indicating the intensity and spatial extent of organic contamination due to disposal of waste from land-based marine fish farms (LBMFFs). Three species of macroalgae from the genus Fucus and the green macroalgae Codium tomentosum were selected for study. The study was carried out at seven flat marine fish farms located in Galicia (NW Spain). Tests were carried out to determine the intra-annual variation in δ¹⁵N values and any differences between selected macroalgae. The δ¹⁵N values enrichment was observed close to the disposal point, and δ¹⁵N values varied more widely throughout the year (±5.57 ‰) at sites affected by the marine fish farm effluent compared to natural conditions (±2 ‰). No significant differences in the isotopic signals were observed in the different species studied (standard major axis). The δ¹⁵N values of macroalgae may be an ideal means of detecting the presence of LBMFFs effluents.     

Synthesis of camptothecin–amino acid carbamate linkers

A more convenient and facile approach for the synthesis and production of camptothecin–amino acids carbamate linkers, that can be used in the synthesis of bioconjugate peptides JF-10-81, JF-10-71, and other peptide analogs designed to target somatostatin receptors has been described.     

NMR determination of pKa values in α-synuclein

The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.     

Foliar δ15N values characterize soil N cycling and reflect nitrate or ammonium preference of plants along a temperate grassland gradient

The natural abundance of stable (15)N isotopes in soils and plants is potentially a simple tool to assess ecosystem N dynamics. Several open questions remain, however, in particular regarding the mechanisms driving the variability of foliar delta(15)N values of non-N(2) fixing plants within and across ecosystems. The goal of the work presented here was therefore to: (1) characterize the relationship between soil net mineralization and variability of foliar Deltadelta(15)N (delta(15)Nleaf - delta(15)Nsoil) values from 20 different plant species within and across 18 grassland sites; (2) to determine in situ if a plant's preference for NO (3) (-) or NH (4) (+) uptake explains variability in foliar Deltadelta(15)N among different plant species within an ecosystem; and (3) test if variability in foliar Deltadelta(15)N among species or functional group is consistent across 18 grassland sites. Deltadelta(15)N values of the 20 different plant species were positively related to soil net mineralization rates across the 18 sites. We found that within a site, foliar Deltadelta(15)N values increased with the species' NO (3) (-) to NH (4) (+) uptake ratios. Interestingly, the slope of this relationship differed in direction from previously published studies. Finally, the variability in foliar Deltadelta(15)N values among species was not consistent across 18 grassland sites but was significantly influenced by N mineralization rates and the abundance of a particular species in a site. Our findings improve the mechanistic understanding of the commonly observed variability in foliar Deltadelta(15)N among different plant species. In particular we were able to show that within a site, foliar delta(15)N values nicely reflect a plant's N source but that the direction of the relationship between NO (3) (-) to NH (4) (+) uptake and foliar Deltadelta(15)N values is not universal. Using a large set of data, our study highlights that foliar Deltadelta(15)N values are valuable tools to assess plant N uptake patterns and to characterize the soil N cycle across different ecosystems.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

Are low cholesterol values associated with excess mortality?

The relation between cholesterol concentration and mortality was studied prospectively over 17 years in 630 New Zealand Maoris aged 25-74. The dead or alive state of each person was determined in 1981. The causes of death were divided into three categories: cancer, cardiovascular disease, and "other." Using univariate and both linear and non-linear multivariate methods of analysis for survivorship data, significant inverse relations with serum cholesterol were found for total mortality in women, for mortality from cancer in men and women, and for other causes of mortality in both men and women. The inverse and non-linear association with total mortality in women remained significant when deaths in the first five years of follow up were excluded. This suggests that the association was not explained by undetected illness causing low cholesterol concentrations at the time of initial examination.