A set of HA-detected experiments for measuring scalar and residual dipolar couplings

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual , and scalar and residual dipolar couplings of ¹⁵N/¹³C labeled proteins in D₂O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making ¹H^N detected experiments unattractive. In addition, the tetrahedral coordination of C is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the and couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of ¹⁵N/¹³C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitins refined solution structure.     

The humus layer determines SO<Subscript>4</Subscript><Superscript>2−</Superscript> isotope values in the mineral soil

Biocycling of sulfur (S) has been proposed to play an important role in the recovery of ecosystems following anthropogenic S deposition. Here, we investigated the importance of the humus layer in the biocycling of S in three forested catchments in the Gårdsjön area of southwestern Sweden with differing S inputs and S isotope signature values. These experimental sites consisted of two reference catchments and the Gårdsjön roof experiment catchment (G1), where anthropogenic deposition was intercepted from 1991 until May 2002 by a roof placed over the entire catchment area. Under the roof, controlled levels of deposition were applied, using a sprinkler system, and the only form of S added was marine SO₄²⁻ with a δ of +19.5‰.We installed ion exchange resin bags at the interface between the humus layer and mineral soil at each of the catchments to collect SO₄²⁻ passing through the humus. The resin bags were installed on four occasions, in 1999 and 2000, covering two summer and two winter periods. The ions collected by each bag during these sampling periods were then eluted and their δ values and SO₄²⁻ concentrations determined. The most striking result is that the average δ value in the resin bags was more than 12‰ lower compared to that of the sprinkler water in the G1 roof catchment. There was no increasing trend in the isotope value in the resin bag SO₄²⁻ despite that the roof treatment has been on-going for almost 10 years; the average value for all resin bags was +7.1‰. The highest δ values found in the G1 roof catchment were between +11‰ and +12‰. However, these values were all obtained from resin bags installed at a single sampling location. Throughfall and resin bag δ values were more similar in the two reference catchments: about +7.5‰ in both cases. There was, however, an increase in resin bag δ values during the first winter period, from about +7‰ to +9‰. The resin bag δ value was linearly and positively related (r² = 0.26, p < 0.001) to the amount of SO₄²⁻ extracted from the resin bags, if relatively high amounts (>50 mmol m⁻²) were excluded. High amounts of resin bag SO₄²⁻ seemed to be related to groundwater inputs, as indicated by the δ value. Our results suggest that rapid immobilization of SO₄²⁻ into a large organic S pool may alter the S isotope value and affect the δ values measured in the mineral soil and runoff.     

Measurement of eight scalar and dipolar couplings for methine–methylene pairs in proteins and nucleic acids

A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for C–C moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C –C groups in a 24-nt RNA oligomer. Chemical shifts of C, C and H (respectively C , C and H ) are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to ¹J_CH, ²J_CH2+²J_CH3, ²J_CH, ¹J_CH2+¹J_CH3, ¹J_CH2–²J_H2H3, ¹J_CH3–²J_H2H3, ³J_HH2 and ³J_HH3 for proteins, and ¹J , ²J J , ²J , ¹J +¹J , ¹J J , ¹J J , ³J and ³J in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine–methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C –C groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the ³J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive ₁ rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C H₂ groups in the loop region of the oligomer, in all cases confirmed by ¹J ^{1} $$" align="middle" border="0"> J , and H resonating downfield of H .Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0175-z.     

Formate binding near the redox-active TyrosineD in Photosystem II: consequences on the properties of TyrD

Formate and phosphate affect substantially the rate of tyrosine D (Tyr_D) oxidation and the stability of the radical Tyr in Photosystem II [Hienerwadel R, Boussac A, Breton J and Berthomieu C (1996) Biochemistry 35: 15447–15460]. This observation prompted us to analyze the influence of formate and phosphate on the environment of Tyr_D using FTIR spectroscopy. The ν (CO) IR mode of Tyr at 1503 cm⁻¹ remains unchanged whatever the buffer used at pH 6 and whether formate is present or not in the sample. Similarly, the main IR mode of reduced Tyr_D remains at ≈1250 cm⁻¹ in all tested conditions. We thus conclude that formate does not modify the hydrogen-bonded interactions of Tyr_D and Tyr with neighbouring D2His189 and D2Gln164. In the Tyr_D-state, an IR mode of formate significantly different from that observed in solution, is detected using ¹³C-formate, showing that formate forms a strong electrostatic interaction within PS II. The presence of formate affects also IR bands that may be assigned to an arginine side chain. Upon Tyr formation, formate does not protonate but its binding interaction weakens. A proton uptake by Mes or phosphate buffer is detected, which is not observed when BisTris is used as a buffer. In these latter conditions, IR bands characteristic of the protonation of a carboxylate group of the protein are detected instead. The present IR data and the recent structural model of the Tyr_D environment proposed by Ferreira KN, Iverson TM, Maghlaoui K, Barber J and Iwata S [(2004) Science 303: 1831–1838], suggest that the proton released upon Tyr formation is shared within a hydrogen bonding network including D2Arg294, and CP47Glu364 and that perturbation of this network by formate – possibly binding near D2Arg294 – substantially affects the properties of Tyr_D.     

High groundwater nitrate concentrations inhibit eutrophication of sulphate-rich freshwater wetlands

During the last 60 years, pollution of the groundwater with has greatly increased in many parts of Europe, as a consequence of excessive use of manure and synthetic fertilisers. Monitoring of groundwater-fed wetlands indicated that sediments with high concentrations had the lowest Fe and concentrations in the pore water. A comparison of two restored open water fens, differing in supply via the groundwater, indicated that the redox potential and the sulphate ( ) reduction rate were lower when the groundwater contained not only but also high concentrations. The lower reduction rates in the -rich open water fen were associated with lower concentrations and the presence of plant species characteristic of clear water. In contrast, the higher reduction rates in the -poor open water fen were associated with very high concentrations and massive development of plant species characteristic of eutrophic environments. Investigations at -rich seepage sites in black alder carrs, showed that high concentrations in the pore water caused chlorosis in the alder carr vegetation, due to lower availability of Fe in the pore water and less Fe uptake by the plants. Experimental desiccation of sediments proved that the -rich seepage sites contained no oxidisable FeS _x , contrary to -poor locations, which became acidified and mobilised extremely high amounts of due to FeS _x oxidation. A laboratory experiment showed that addition to sediments led to reduced releases of Fe, and S^2–, very likely due to the oxidation of reduced Fe and S compounds. Overall, the results confirmed that is an energetically more favourable electron acceptor in anaerobic sediments than Fe and , and that high loads function as a redox buffer, preventing reduction of Fe and . Limited reduction prevents S^2– -mediated mobilisation of from Fe- complexes. At a higher redox potential, reduced Fe, including FeS _x , was oxidised, increasing the content of Fe(III) capable of binding . This prevented increased availability and the concomitant massive development of plant species characteristic of eutrophic environments.     

Altered expression of an ankyrin-repeat protein results in leaf abnormalities,necrotic lesions,and the elaboration of a systemic signal

Summary The PR-like proteins, class I -1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated lucanohydrolase inding roteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved lant-specific yrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

An evolutionary analytical model of a complementary circular code simulating the protein coding genes, the 5′ and 3′ regions

The self-complementary subset ∪{AAA,TTT} with = {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} of 22 trinucleotides has a preferential occurrence in the frame 0 (reading frame established by the ATG start trinucleotide) of protein (coding) genes of both prokaryotes and eukaryotes. The subsets ∪{CCC} and ∪{GGG} of 21 trinucleotides have a preferential occurrence in the shifted frames 1 and 2 respectively (frame 0 shifted by one and two nucleotides respectively in the 5′-3′ direction). and are complementary to each other. The subset contains the subset which has the rarity property (6 × 10⁻⁸) to be a complementary maximal circular code with two permutated maximal circular codes and in the frames 1 and 2 respectively. is called a C³ code. A quantitative study of these three subsets in the three frames 0, 1, 2 of protein genes, and the 5′ and 3′ regions of eukaryotes, shows that their occurrence frequencies are constant functions of the trinucleotide positions in the sequences. The frequencies of in the frame 0 of protein genes are 49, 28.5 and 22.5% respectively. In contrast, the frequencies of in the 5′ and 3′ regions of eukaryotes, are independent of the frame. Indeed, the frequency of in the three frames of 5′ (respectively 3′) regions is equal to 35.5% (respectively 38%) and is greater than the frequencies and , both equal to 32.25% (respectively 31%) in the three frames. Several frequency asymmetries unexpectedly observed (e.g. the frequency difference between and in the frame 0), are related to a new property of the subset involving substitutions. An evolutionary analytical model at three parameters (p, q, t) based on an independent mixing of the 22 codons (trinucleotides in frame 0) of with equiprobability (1/22) followed by t ≈ 4 substitutions per codon according to the proportions p ≈ 0.1; q ≈ 0.1 and r = 1 − p − q ≈ 0.8 in the three codon sites respectively, retrieves the frequencies of observed in the three frames of protein genes and explains these asymmetries. Furthermore, the same model (0.1, 0.1, t) after t ≈ 22 substitutions per codon, retrieves the statistical properties observed in the three frames of the 5′ and 3′ regions. The complex behaviour of these analytical curves is totally unexpected and a priori difficult to imagine.     

Compensatory changes in Photosystem II electron turnover rates protect photosynthesis from photoinhibition

Exposure of algae or higher plants to bright light can result in a photoinhibitory reduction in the number of functional PS II reaction centers (n) and a consequential decrease in the maximum quantum yield of photosynthesis. However, we found that light-saturated photosynthetic rates (P_max) in natural phytoplankton assemblages sampled from the south Pacific ocean were not reduced despite photoinhibitory decreases in n of up to 52%. This striking insensitivity of P_max to photoinhibition resulted from reciprocal increases in electron turnover ( )through the remaining functional PS II centers. Similar insensitivity of P_max was also observed in low light adapted cultures of Thalassiosira weissflogii (a marine diatom), but not in high light adapted cells where P_max decreased in proportion to n. This differential sensitivity to decreases in n occurred because was close to the maximum achievable rate in the high light adapted cells, whereas was initially low in the low light adapted cells and could thus increase in response to decreases in n. Our results indicate that decreases in plant productivity are not necessarily commensurate with photoinhibition, but rather will only occur if decreases in n are sufficient to maximize or incident irradiance becomes subsaturating.     

Estimating the Accuracy of Protein Structures using Residual Dipolar Couplings

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone ¹H–¹⁵N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Sulfur isotope values in a forested catchment over four years: Evidence for oxidation and reduction processes

A small catchment on the Swedish West Coast has been studied over four years to determine S dynamics by using S isotope ratios. A Norway spruce dominated forest covers the catchment, and small peat areas occur in the lower parts of the catchment. The runoff values varied both during the year, and from year to year. Over the period from February 1990 to December 1993, the values ranged from — 1%. to +11%. Over the same period, the throughfall values ranged from +1%. to +15%. There was no correlation (r ²= 0.01; Pr(F)=0.57) between values in throughfall and runoff. Since the only input of S to the catchment is atmospheric deposition, the long-term runoff S mass flux is controlled by the deposition. Therefore, processes in the catchment are responsible for the variation in the runoff values. During periods with enriched runoff, bacterial dissimilatory SO ₄ ^2– reduction occurs in the catchment. After very dry periods, oxidation of this reduced S, which is³²S-enriched, can be traced in runoff. Previous studies of the catchment have not been able to distinguish between: 1) oxidation of reduced S and dry deposition, and 2) reduction and adsorption. From the current study, it can be concluded that adsorption and dry deposition cannot cause the observed variation in runoff .     

Regulation of mineral nitrogen uptake in plants

In the biosphere plants are exposed to different forms of N, which comprise mineral and organic N forms in soils as well as gaseous NH3, NOx, and molecular N2 in the atmosphere. The form of N uptake is mainly determined by its abundance and accessibility, which make and the most important N forms for plant nutrition under agricultural conditions. With minor importance, the form of N uptake is also subject to plant preferences, by which plants maintain their cation/anion balance during uptake. However, some species seem to have an obligatory preference which even prevents their growth on certain other N sources. In general, uptake of a certain N form closely matches the growth-related demand of the plant, at least when N transport to the root surface is not limiting. In addition, many plants accumulate large pools of N during vegetative growth which are remobilized in the generative stage. As a consequence, systems responsible for N transport need to be tightly regulated in their expression and activity upon sensing N availability and plant demand. Employing the tools of molecular genetics, the first plant genes encoding transporters for inorganic N have recently been isolated and characterized. These data can now complete the wealth of physiological and nutritional studies on N uptake. The present article will focus on the uptake of and into root cells and tries to link data derived from physiological, genetic and molecular studies.     

A new medium formulation for <Emphasis Type="Italic">in vitro</Emphasis> rooting of carob tree based on leaf macronutrients concentrations

Experiments were performed to optimize the macronutrients concentrations for in vitro rooting of Ceratonia siliqua micropropagated shoots. Several dilutions of Murashige and Skoog (MS) medium were tested: full-strength MS, half-strength MS ( MS), and MS + full N. The frequency of in vitro rooting was enhanced when the MS was used (50 % rooted shoots). Mature leaves from 20 – 30 year-old carob trees and from 2 year-old micropropagated plants were collected and the concentrations of macronutrients (N, P, K, Ca, Mg) assessed. Based on the mineral composition of the leaves a new medium was formulated and compared with the previous ones showing an increment of the rooting frequency to 80 %. Moreover, shoots rooted in the new medium did not show symptoms of apical necrosis that occurred in the other tested media.     

Water relations of buried eggs of mound building birds

Summary Eggs of the brush turkey (BT) and mallee fowl (MF) are incubated in mounds of soil and plant litter. Humidity in BT mounds is always near saturation (>99% RH), but in MF mounds it drops to lower values in summer (x=77% RH). Despite these high humidities, the eggs lose an average of 9.5% (BT) and 12.0% (MF) of their initial mass by evaporation before hatching. The rate of evaporation increases during incubation several-fold due to large changes in water vapor conductance of the shell and embryonic heat production. Values of in fully incubated eggs in mound material are about 3–6 times higher than values obtained from unincubated eggs in desiccators. This effect depends on two factors: (1) increases with ambient humidity, especially above 80% RH, possibly because the effective site of evaporation moves out along the walls of the pores in the eggshell. (2) Structural changes of the pores due to calcium absorption by the embryo directly increase . The first factor is most important in BT eggs and the second is dominant in MF eggs. Production of metabolic heat by the embryo increases the vapor pressure difference across the shell and further increases , especially in mounds of high humidity. The changes in pore structure are adaptive because they produce high conductances to respiratory gases and cause normal gas tensions within the egg at the end of development, yet is low enough in early development to prevent excessive water loss. Water not lost by evaporation or taken up by the embryo is stored and released during hatching. A small amount of mass is lost during incubation by respiratory gas exchange.Abbreviations BT brush turkey - MF mallee fowl - RH relative humidity     

(H)NCAHA and (H)CANNH experiments for the determination of vicinal coupling constants related to the ϕ-torsion angle

Summary A set of three-dimensional triple-resonance experiments is described which provide , , and coupling constants. The pulse sequences generate E.COSY-like multiplet patterns and comprise a magnetization transfer from the amide proton to the α-proton or vice versa via the directly bound heteronuclei. For residues with the ¹H^α spin resonating close to the H₂O signal, a modified HNCA experiment can be employed to measure the vicinal ¹H^N,¹H^α couplings. Ambiguities associated with the conversion of values into ϕ-angle constraints for protein structure determination can be resolved with the knowledge of the heteronuclear ³J-couplings. In favourable cases, stereospecific assignments of glycine α-protons can be obtained by employing the experiments described here in combination with NOE data. The methods are applied to flavodoxin from Desulfovibrio vulgaris.     

Changes in Swallowing-Related Tachycardia and Respiratory Arrhythmia Induced by Modulation of Tonic Parasympathetic Influences

In 19 tested persons in the resting lying position, we examined changes in the mean duration of R-R intervals and in parasympathetic chronotropic cardiac reactions (respiratory arrhythmia,RA, and swallowing-related tachycardia, ST) induced by intensified tonic parasympathetic influences after peroral administration of antagonists of muscarinic (M) receptors in small doses. Administration of 0.02 g of an extract of Belladonna, which contains alkaloids of the atropine group, led to a significant increase (P < 0.001). In this case, the RA at the natural frequency of breathing (P < 0.05) and ST (P < 0.001) simultaneously increased. Close correlation was observed both between initial values of the ST and in various tested persons (r = 0.87) and between changes in these values, which were induced by the action of small amounts of M antagonists (r = 84). In various persons, the RA during deep slowed breathing (6 min^-1, RA₆) demonstrated ambiguous modifications. In the structure of RA₆, we identified two components: an inspiratory tachycardic (TC) and an expiratory bradycardic (BC). With intensification of the parasympathetic influences, the TC began to prevail; its increase was strongly related to a rise in the (r = 0.84). In contrast, correlation between changes in the BC and an increase in the was completely absent (r = 0.001). Therefore, small amounts of M antagonists intensify both the tonic component of parasympathetic control (increasing the ) and its dynamic indices, the magnitudes of the ST and RA. Tachycardia during swallowing and the inspiratory TC under conditions of deep slowed breathing are closely related to changes in the parasympathetic influences. The magnitude of the RA is the index, which reflects the tonic component of chtonotropic control under conditions of both slowed and natural breathing less adequately than the ST.     

13C spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments

A set of new NMR pulse sequences has been designed for the measurement of ¹³C relaxation rate constants in RNA and DNA bases: the spin-lattice relaxation rate constant R(C_z), the spin-spin relaxation rate constant R(C₊), and the CSA-dipolar cross-correlated relaxation rate constant . The use of spin-state selective correlation techniques provides increased sensitivity and spectral resolution. Sensitivity optimised C-C filters are included in the pulse schemes for the suppression of signals originating from undesired carbon isotopomers. The experiments are applied to a 15% ¹³C-labelled 33-mer RNA–theophylline complex. The measured ratios indicate that ¹³C CSA tensors do not vary significantly for the same type of carbon (C₂, C₆, C₈), but that they differ from one type to another. In addition, conformational exchange effects in the RNA bases are detected as a change in the relaxation decay of the narrow ¹³C doublet component when varying the spacing of a CPMG pulse train. This new approach allows the detection of small exchange effects with a higher precision compared to conventional techniques.     

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to ¹³C and ¹⁵N resonances on the nucleotide base side chains. We report here the application of the alternate-site ¹³C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing ¹³C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to interaction in T ₁ measurements of larger nucleic acids and in T _1ρ measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.     

Chronic nitrate additions dramatically increase the export of carbon and nitrogen from northern hardwood ecosystems

A long-term field experiment was initiated to simulate chronic atmospheric N deposition, a widespread phenomenon in industrial regions of the world. Eight years of experimental nitrate ( ) additions (3 g -N m^–2 per year) to four different northern hardwood forests located along a 500 km geographic gradient dramatically increased leaching losses of -N, dissolved organic carbon (DOC), and dissolved organic nitrogen (DON). During the last two water years, the average increase in solution -N and DON leaching from the -amended plots was 2.2 g N m^–2, equivalent to 72% of the annual experimental N addition. Results indicate that atmospheric N deposition may rapidly saturate some northern hardwood ecosystems across an entire biome in the upper Great Lakes Region of the USA. Changes in soil C and N cycling induced by chronic N deposition have the potential in this landscape to significantly alter the flux of DOC and DON from upland to aquatic ecosystems. Michigan Gradient study site characteristics are similar to those of European forests most susceptible to N saturation.