Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.     

Stable isotope-based statistical tools as ecological indicator of pollution sources in Mediterranean transitional water ecosystems

N-stable isotope analysis of macroalgae has become a popular method for the monitoring of nitrogen pollution in aquatic ecosystems. Basing on changes in their δ¹⁵N, macroalgae have been successfully used as biological traps to intercept nitrogen inputs. As different nitrogen sources differ in their isotopic signature, this technique provides useful information on the origin of pollutants and their extension in the water body. However, isotopic fractionation potentially resulting from microbial nitrogen processing, and indirect isotopic variations due to effects of physicochemical conditions on algal nutrient uptake and metabolism, may affect anthropogenic N isotopic values during transportation and assimilation. This in turn can affect the observed isotopic signature in the algal tissue, inducing isotopic variations not related to the origin of assimilated nitrogen, representing a “background noise” in isotope-based water pollution studies.In this study, we focused on three neighbouring coastal lakes (Caprolace, Fogliano and Sabaudia lakes) located south of Rome (Italy). Lakes were characterized by differences in terms of anthropogenic pressure (i.e. urbanization, cultivated crops, livestock grazing) and potential “background noise” levels (i.e. nutrient concentration, pH, microbial concentration). Our aim was to assess nitrogen isotopic variations in fragments of Ulva lactuca specimens after 48 h of submersion to identify and locate the origins of nitrogen pollutants affecting each lake. δ¹⁵N were obtained for replicated specimens of U. lactuca spatially distributed to cover the entire surface of each lake, previously collected from a benchmark, unpolluted site. In order to reduce the environmental background noise on isotopic observations, a Bayesian hierarchical model relating isotopic variation to environmental covariates and random spatial effects was used to describe and understand the distribution of isotopic signals in each lake.Our procedure (i) allowed to remove background noise and confounding effects from the observed isotopic signals; (ii) allowed to detect “hidden” pollution sources that would not be detected when not accounting for the confounding effect of environmental background noise; (iii) produced maps of the three lakes providing a clear representation of the isotopic signal variation even where background noise was high. Maps were useful to locate nitrogen pollution sources, identify the origin of the dissolved nitrogen and quantify the extent of pollutants, showing localized organic pollution impacting Sabaudia and Fogliano, but not Caprolace. This method provided a clear characterization of both intra- and inter-lake anthropogenic pressure gradients, representing a powerful approach to the ecological indication and nitrogen pollution management in complex systems, as transitional waterbodies are.     

A triple-resonance pulse scheme for selectively correlating amide1HN and15N nuclei with the1Hα proton of the preceding residue

Summary A 3D¹H–¹⁵N–¹³C triple resonance experiment is presented that contains exclusively cross peaks between the¹H^N and¹⁵N nuclei of one residue with the H of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T₂ values. The experiment consists of coherence transfers via one-bond couplings from the H^N via N, CO, C to the H and back to the H^N for detection; it is called HN(COCA)HA. The experiment was tested on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.     

Evaluation of the availability ofAzolla-N and urea-N to rice using15N

Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha^–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two¹⁵N methods. In the first,Azolla caroliniana (Willd.) was labelled with¹⁵N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha^–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of¹⁵N-urea was applied separately or in combination with either 250 or 330 kg N ha^–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha^–1 to 430 kg N ha^–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using¹⁵N-labelledAzolla, this method is preferable to the¹⁵N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla.     

Competition between siratro and kleingrass for15N labelled mineralized nitrogen

Summary Isotope dilution provides a method for measuring plant competition for mineral N and transfer of biologically fixed N from a legume to a grass. A plant growth medium was enriched with¹⁵N, and used to grow Siratro (Macropitilium atropurpureum D.C. Urb.) and Kleingrass 75 (Panicum coloratum L.) in 20 liter pots for 98 days in a glasshouse. The plants were grown in pure stand and in mixtures. When grown in 50∶50 mixture the grass obtained 59% of the labelled N and the legume obtained 41%. The grass produced nearly as much root mass as the legume even though biomass of the shoots were less than half that of the legume. Reducing the proportion of either plant species in the mixture reduced the proportion of the mineralized N absorbed by that species. The shoots of the grass were significantly more enriched (1.166 atom%¹⁵N excess) than the roots (1.036). The grass received 12% of its N as biologically fixed N from the legume.     

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)