Comparative <Superscript>1</Superscript>H NMR-based metabonomic analysis of HIV-1 sera

¹H NMR spectroscopy of sera from HIV-1 infected and uninfected individuals was performed on 300 and 600 MHz instruments. The resultant spectra were automatically data reduced to 90 and 180 integral segments of equal length. Analysis of variance identified significant differences between the sample groups, especially for the samples analyzed on 600 MHz and reduced to fewer segments. Linear discriminant analysis correctly classified 100% of the samples analyzed on the 300 MHz NMR (reduced to 180 segments); an increase in instrument sensitivity resulted in lower percentages of correctly classified samples. Multinomial logistic regression (MLR) resulted in 100% correct classification of all samples from both instruments. Thus ¹H-NMR metabonomics on either instrument distinguishes HIV-positive individuals using or not using anti retroviral therapy, but the sensitivity of the instrument impacts on data reduction. Furthermore, MLR is a novel multivariate statistical technique for improved classification of biological data analyzed in NMR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

HNHC: a triple resonance experiment for correlating the H2, N1(N3) and C2 resonances in adenine nucleobases of <Superscript>13</Superscript>C-, <Superscript>15</Superscript>N-labeled RNA oligonucleotides

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in adenine nucleobases of ¹³C, ¹⁵N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a ²J-¹⁵N-HSQC and a ¹J-¹³C-HSQC and utilizes large ²J(H2, N1(N3)) and ¹J(H2, C2) couplings. The experiment was applied to a medium-size ¹³C, ¹⁵N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the ¹H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR 20:173–176 2001) is provided. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Empirical correlation between protein backbone <Superscript>15</Superscript>N and <Superscript>13</Superscript>C secondary chemical shifts and its application to nitrogen chemical shift re-referencing

The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting ¹³C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not require extensive assignment of the NMR data. We report here a way of extending the LACS approach to ¹⁵N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for this nucleus. The approach is based on our finding that the secondary ¹⁵N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and beta carbons of residue i − 1. Thus once alpha and beta ¹³C chemical shifts are available (their difference is referencing error-free), the ¹⁵N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior to a structure determination and can be used to analyze potential referencing problems in database data not associated with three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed that nearly 35% of the BMRB entries have δ ¹⁵N values mis-referenced by over 0.7 ppm and over 25% of them have δ ¹H^N values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone ¹⁵N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>13</Superscript>C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a ¹³CH₃-reporter attached. This ¹³C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of EDTA to minimize ionic strength dependent frequency shifts in the <Superscript>1</Superscript>H NMR spectra of urine

The ¹H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). ¹H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d₄, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>1</Superscript>H NMR metabolic fingerprinting of saffron extracts

The aim of this study was to explore feasibility of ¹H NMR metabolic fingerprinting for discrimination of authenticity of saffron using principal component analysis (PCA) modeling. Authentic reference Iranian saffron (n = 31) and commercial samples (n = 32) were used. Cross-validated PCA models based on ¹H NMR spectra of solutions prepared by direct extraction of grinded saffron with methanol-d ₄ distinguished reference Iranian saffron samples from commercial samples that formed several distinct clusters, some of which represent falsified samples as confirmed by microscopic analysis. The production sites and drying conditions of the authentic reference Iranian samples were not reflected in the current dataset. Picrocrocin and glycosyl esters of crocetin emerged as the most important ¹H NMR markers of authentic saffron by using statistical correlation spectroscopy. In conclusion, ¹H NMR spectra of saffron extracts combined with pattern recognition by PCA provide immediate means of unsupervised classification of saffron samples.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.     

Deuterium isotope effects on <Superscript>15</Superscript>N backbone chemical shifts in proteins

Quantum mechanical calculations are presented that predict that one-bond deuterium isotope effects on the ¹⁵N chemical shift of backbone amides of proteins, ¹Δ¹⁵N(D), are sensitive to backbone conformation and hydrogen bonding. A quantitative empirical model for ¹Δ¹⁵N(D) including the backbone dihedral angles, Φ and Ψ, and the hydrogen bonding geometry is presented for glycine and amino acid residues with aliphatic side chains. The effect of hydrogen bonding is rationalized in part as an electric-field effect on the first derivative of the nuclear shielding with respect to N–H bond length. Another contributing factor is the effect of increased anharmonicity of the N–H stretching vibrational state upon hydrogen bonding, which results in an altered N–H/N–D equilibrium bond length ratio. The N–H stretching anharmonicity contribution falls off with the cosine of the N–H···O bond angle. For residues with uncharged side chains a very good prediction of isotope effects can be made. Thus, for proteins with known secondary structures, ¹Δ¹⁵N(D) can provide insights into hydrogen bonding geometries. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

<Superscript>13</Superscript>C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of ¹³C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose ¹³C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of ¹³C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, ¹³C-direct detected NMR methods constitute useful complements to the conventional ¹H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.     

Identification of a plant-specific Zn<Superscript>2+</Superscript>-sensitive ribonuclease activity

Ribonucleases (RNases) play a variety of cellular and biological roles in all three domains of life. In an attempt to perform RNA immuno-precipitation assays of Arabidopsis proteins, we found an EDTA-dependent RNase activity from Arabidopsis suspension tissue cultures. Further investigations proved that the EDTA-dependent RNase activity was plant specific. Characterization of the RNase activity indicated that it was insensitive to low pH and high concentration of NaCl. In the process of isolating the activity with cation exchange chromatography, we found that the EDTA dependency of the activity was lost. This led us to speculate that some metal ions, which inhibited the RNase activity, may be removed during cation exchange chromatography so that the nuclease activity was released. The EDTA dependency of the activity could be due to the ability of the EDTA chelating those metal ions, mimicking the effect of the cation exchange chromatography. Indeed, Zn²⁺ strongly inhibited the activity, and the inhibition could be released by EDTA based on both in-solution and in-gel assays. In-gel assays identified two RNase activity bands. Mass spectrometry assays of those activity bands revealed more than 20 proteins. However, none of them has an apparent known nuclease domain, suggesting that one or more of those proteins might possess a currently uncharacterized nuclease domain. Our results may shed light on RNA metabolism in plants by introducing a novel plant-specific RNase activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively <Superscript>13</Superscript>C-labelled proteins

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-¹³C]- and [2-¹³C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [¹³C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in ¹³C-¹³C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cancer/testis antigens can be immunological targets in clonogenic CD133<Superscript>+</Superscript> melanoma cells

“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133⁺ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133⁺ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8⁺ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Statistical analysis of structural characteristics of protein Ca<Superscript>2+</Superscript>-binding sites

To better understand the biological significance of Ca²⁺, we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca²⁺-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 ± 2 vs. 7 ± 1), fewer protein ligands (4 ± 2 vs. 6 ± 1), and more water ligands (2 ± 2 vs. 1 ± 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H₂O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H₂O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 ± 1 vs. 3 ± 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix–loop–helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca–O–C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca²⁺-binding proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Michael Kirberger, Xue Wang, and Hai Deng contributed equally to this article.     

Solution structure of tRNA<Superscript>Val</Superscript> from refinement of homology model against residual dipolar coupling and SAXS data

A procedure is presented for refinement of a homology model of E. coli tRNA^Val, originally based on the X-ray structure of yeast tRNA^Phe, using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N–H^N RDCs measured with Pf1 phage alignment, and 20 imino N–H^N RDCs obtained from magnetic field dependent alignment of tRNA^Val. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA^Phe by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q _free = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA^Phe, in agreement with previous NMR-based tRNA^Val models. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Breast cancer cells expressing stem cell markers CD44<Superscript>+</Superscript> CD24<Superscript>lo</Superscript> are eliminated by Numb-1 peptide-activated T cells

Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen⁺ (ESA) CD44^hi CD24^lo, CD44^hi CD133⁺, and CD133⁺ CD24^lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM^Res MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM^Sens MCF7. The levels of Numb, and Numb-L-[P]-Ser²⁶⁵ were similar in GEM^Res and GEM^Sens MCF7 cells. Only the levels of Numb-L (long)-Ser²⁹⁵ decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEM^Res MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112–2120) and Numb-1 (87–95) eliminated NICD^positive, CD24^hi CD24^lo MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative analysis of the orientation of transmembrane peptides using solid-state <Superscript>2</Superscript>H- and <Superscript>15</Superscript>N-NMR: mobility matters

Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: ²H-NMR quadrupolar waves, and separated local field ¹⁵N–¹H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by ²H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.     

Natural Abundance of <Superscript>15</Superscript>N in Nitrogen-Limited Forests and Tundra Can Estimate Nitrogen Cycling Through Mycorrhizal Fungi: A Review

The hyphae of ectomycorrhizal and ericoid mycorrhizal fungi proliferate in nitrogen (N)-limited forests and tundra where the availability of inorganic N is low; under these conditions the most common fungal species are those capable of protein degradation that can supply their host plants with organic N. Although it is widely understood that these symbiotic fungi supply N to their host plants, the transfer is difficult to quantify in the field. A novel approach uses the natural ¹⁵N:¹⁴N ratios (expressed as δ¹⁵N values) in plants, soils, and mycorrhizal fungi to estimate the fraction of N in symbiotic trees and shrubs that enters through mycorrhizal fungi. This calculation is possible because mycorrhizal fungi discriminate against ¹⁵N when they create compounds for transfer to plants; host plants are depleted in ¹⁵N, whereas mycorrhizal fungi are enriched in ¹⁵N. The amount of carbon (C) supplied to these fungi can be stoichiometrically calculated from the fraction of plant N derived from the symbiosis, the N demand of the plants, the fungal C:N ratio, and the fraction of N retained in the fungi. Up to a third of C allocated belowground, or 20% of net primary production, is used to support ectomycorrhizal fungi. As anthropogenic N inputs increase, the C allocation to fungi decreases and plant δ¹⁵N increases. Careful analyses of δ¹⁵N patterns in systems dominated by ectomycorrhizal and ericoid mycorrhizal symbioses may reveal the ecosystem-scale effects of alterations in the plant–mycorrhizal symbioses caused by shifts in climate and N deposition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.