Partial solid-state NMR <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of a perdeuterated back-exchanged seven-transmembrane helical protein <Emphasis Type="Italic">Anabaena</Emphasis> Sensory Rhodopsin

Anabaena Sensory Rhodopsin (ASR) is a unique photochromic membrane-embedded photosensor which interacts with soluble transducer and is likely involved in a light-dependent gene regulation in the cyanobacterium Anabaena sp. PCC 7120. We report partial spectroscopic ¹H, ¹³C and ¹⁵N assignments of perdeuterated and back-exchanged ASR reconstituted in lipids. The reported assignments are in general agreement with previously determined assignments of carbon and nitrogen resonances in fully protonated samples. Because the back-exchange was performed on ASR in a detergent-solubilized state, the location of detected residues reports on the solvent accessibility of ASR in detergent. A comparison with the results of previously published hydrogen/exchange data collected on the ASR reconstituted in lipids, suggests that the protein has larger solvent accessible surface in the detergent-solubilized state.     

Stereospecific complexation of uranyl ion with tartaric acids studied by NMR spectroscopy

A full pH range ¹H and ¹³C NMR study was performed on the complexation of UO₂²⁺ with (D,L)- and meso-tartaric acids, for variable concentrations and molar ratios, in comparison with (D)-tartaric acid. The main result is that, in spite of the already high number of complexes formed with the active ligand, an additional species occurs with the racemic mixture for which experimental evidence indicates a cyclic trimer structure. A smaller number of complexes is formed with meso-tartaric acid. Information on the conformation of bound ligand is also obtained.     

Greenhouse evaluation of fitness-related reproductive traits in roundup<Superscript>®</Superscript>-tolerant transgenic creeping bentgrass (<Emphasis Type="Italic">Agrostis Stolonifera</Emphasis> L.)

Summary Creeping bentgrass is a very important turfgrass species used extensively on golf course greens, fairways, and tees. One of the challenges of creeping bentgrass management is the control of grassy weeds, most of which respond to herbicides in a similar manner to that of creeping bentgrass. As part of a weed management program for golf courses, Roundup^?-tolerant creeping bentgrass will be simple to employ and more effective in controlling problem weeds than currently available methods. The goal of this research was to evaluate fitness-related reproductive traits in four transgenic creeping bentgrass events modified to express a Roundup^?-tolerant gene, cp4 epsps, to determine if these creeping bentgrass events had gained an unexpected reproductive fitness advantage. We compared transgenic events ASR 333, ASR801 with their nontransformed tissue culture line, C99056L and transgenic events ASR365, ASR368 with their non-transformed tissue culture line, B99061R. Populations of plants from three conventional cultivars were also included for comparison to determine whether significant variations, if present in transgenic events, were novel to the non-transformed organism, Agrostis stolonifera L. Our results showed that none of the four transgenic events surveyed were significantly different from the respective non-transformed tissue culture line plants for the following characteristics: first heading date, anthesis duration, inflorescence length, number of florets per inflorescence, pollen size, and seed-set capacity through open-pollination. One of the transgenic events, ASR333, needed significantly more days for anthesis initiation than the nontransformed tissue culture line, C99056L; while another transgenic event, ASR801, exhibited significantly shorter pollen longevity than plants of the tissue culture line, C99056L. However, ASR801 was not significantly different from the conventional cultivars ‘Penn A-4’ and ‘Penncross’ for pollen longevity. Plants of both transgenic events ASR365 and ASR368 did not differ significantly from plants of the tissue culture line, B99061R, for all characters measured.     

COMPLETE-MFA: Complementary parallel labeling experiments technique for metabolic flux analysis

We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-¹³C], [2-¹³C], [3-¹³C], [4-¹³C], [5-¹³C], and [6-¹³C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography–mass spectrometry (GC–MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution ¹³C-flux analysis.     

Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from ¹³C{¹⁹F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-¹³C]lysine and complexed with desleucyl [¹⁹F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The ¹³C{¹⁹F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.     

Effect of Pre-Stressing on the Acid-Stress Response in Bifidobacterium Revealed Using Proteomic and Physiological Approaches

Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR), caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR), and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH₃, thiols, and peptidoglycan, the activity of H⁺-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing.     

Tomato ABSCISIC ACID STRESS RIPENING (ASR) Gene Family Revisited

Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.     

1H, 13C and 15N backbone and side-chain chemical shift assignments for oxidized and reduced desulfothioredoxin

Based on sequence homology, desulfothioredoxin (DTrx) from Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thioredoxin superfamily. Desulfothioredoxin (104 amino acids) contains a particular active site consensus sequence, CPHC probably correlated to the anaerobic metabolism of these bacteria. We report the full ¹H, ¹³C and ¹⁵N resonance assignments of the reduced and the oxidized form of desulfothioredoxin (DTrx). 2D and 3D heteronuclear NMR experiments were performed using uniformly ¹⁵N-, ¹³C-labelled DTrx. More than 98% backbone and 96% side-chain ¹H, ¹³C and ¹⁵N resonance assignments were obtained. (BMRB deposits with accession number 16712 and 16713).     

Factors That Differentiate the H-bond Strengths of Water Near the Schiff Bases in Bacteriorhodopsin and Anabaena Sensory Rhodopsin

Bacteriorhodopsin (BR) functions as a light-driven proton pump, whereas Anabaena sensory rhodopsin (ASR) is believed to function as a photosensor despite the high similarity in their protein sequences. In Fourier transform infrared (FTIR) spectroscopic studies, the lowest O-D stretch for D₂O was observed at ∼2200 cm⁻¹ in BR but was significantly higher in ASR (>2500 cm⁻¹), which was previously attributed to a water molecule near the Schiff base (W402) that is H-bonded to Asp-85 in BR and Asp-75 in ASR. We investigated the factors that differentiate the lowest O-D stretches of W402 in BR and ASR. Quantum mechanical/molecular mechanical calculations reproduced the H-bond geometries of the crystal structures, and the calculated O-D stretching frequencies were corroborated by the FTIR band assignments. The potential energy profiles indicate that the smaller O-D stretching frequency in BR originates from the significantly higher pK_a(Asp-85) in BR relative to the pK_a(Asp-75) in ASR, which were calculated to be 1.5 and −5.1, respectively. The difference is mostly due to the influences of Ala-53, Arg-82, Glu-194–Glu-204, and Asp-212 on pK_a(Asp-85) in BR and the corresponding residues Ser-47, Arg-72, Ser-188-Asp-198, and Pro-206 on pK_a(Asp-75) in ASR. Because these residues participate in proton transfer pathways in BR but not in ASR, the presence of a strongly H-bonded water molecule near the Schiff base ultimately results from the proton-pumping activity in BR.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

1H, 13C and 199Hg NMR characteristics of new amine-mercuric chloride complexes

Reduction of some N-alkylimines has been achieved with NaBH₄ to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their ¹H and ¹³C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl₂L₂ complexes. The ¹H, ¹³C and ¹⁹⁹Hg NMR chemical shifts have been obtained as well as ¹J(¹³CH) and ²J(¹³CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the ¹⁹⁹Hg.     

Putative Effect of Aquifer Recharge on the Abundance and Taxonomic Composition of Endemic Microbial Communities

Drought events and the overexploitation of freshwater resources have led to the increased need to manage groundwater reserves. Aquifer storage and recovery (ASR), whereby artificial water is injected into aquifers for storage, is one of the proposed methods by which freshwater supplies can be increased. Microbial clogging following injection, however, is a major issue. Here, during laboratory simulations of ASR, we used flow cytometry and bar-coded pyrosequencing to investigate changes in microbial abundance and community dynamics. Bacterial abundance ranged from 5.0 × 10⁴ to 1.4 × 10⁷ cells ml^-1 before the addition of synthetic wastewater. Following wastewater addition, a 25-fold decrease in abundance was observed, coinciding with a 12-fold increase in viral abundance. Taxa shifted from an overrepresentation of Sphingomonadales, Sphingobacteriales, Rhodospirillales, Caulobacterales, Legionellales, Bacillales, Fusobacteriales and Verrucomicrobiales prior to the addition of synthetic wastewater to Burkholderiales, Actinomycetales, Pseudomonadales, Xanthomonadales, Rhodobacterales, Thizobiales and Thiotrichales following the addition of synthetic wastewater. Furthermore, a significant difference in overall taxonomic composition between the groundwater samples before and after the addition of synthetic wastewater was observed, with water samples exhibiting more similarity to sediment samples after wastewater was added. Collectively, these results suggest that ASR may alter the taxonomic composition of endemic microbial communities and that complete profiles of groundwater properties, including microbial community abundance and composition need to be taken into consideration when selecting aquifers for ASR practices.     

Effect of water regime on carbon isotope composition of lichens

δ¹³C values of the lichens Ramalina duriaei and Teloschistes villosus collected in their natural habitat were repeatedly measured during 2 years. Results show variations in the stable carbon isotope ratios (¹³C/¹²C). Such variations are correlated to the seasonal rainfall, i.e. low values of δ¹³C of the lichens during the winter and high values of δ¹³C during the dry summer. Relatively low δ¹³C values were obtained also in laboratory experiments with lichens grown under controlled humid conditions and in lichens collected from humid habitats.     

Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.     

δ13C as a marker to study digesta passage kinetics in ruminants: a combined in vivo and in vitro study

The aim of the current study was to explore the use of the tracer ¹³C as an internal marker to assess feed fraction-specific digesta passage kinetics through the digestive tract of dairy cows. Knowledge on feed-specific fractional passage rates is essential to improve estimations on the extent of rumen degradation and microbial protein efficiency; however, this information is largely lacking. An in vivo and in vitro experiment was conducted with grass silages (Lolium perenne L.) that were enriched with ¹³C by growing the grass under elevated ¹³CO₂ conditions. In a crossover design, two dairy cows received pulse doses of two ¹³C-enriched grass silages and chromium-mordanted neutral detergent fibre (Cr-NDF) into the rumen. The two ¹³C-enriched grass silages used differed in digestibility and were grown under identical field conditions as the bulk silages fed to the animals. Faecal excretion patterns of ¹³C-enriched dry matter (¹³C-DM), neutral detergent fibre (¹³C-NDF) and Cr-NDF were established, and a nonlinear multicompartmental model was used to determine their rumen passage kinetics. In addition, the ¹³C-enriched silages were incubated in rumen liquid in an in vitro batch culture system at different time intervals to determine the effect of fermentation on ¹³C-enrichment in the residue. The in vitro study showed that the ¹³C : ¹²C ratios in DM and NDF residues remained stable from 24 h of incubation onwards. In addition, in vitro fractional degradation rates for ¹²C in the DM and NDF did not differ from those of ¹³C, indicating that fermentative degradation does not affect the ¹³C : ¹²C ratio in the DM nor in the NDF fraction of the residue. Model fits to the faecal excretion curves showed a significant difference in fractional rumen passage rates between Cr-NDF, ¹³C-DM and ¹³C-NDF (P ⩽ 0.025). Silage type had no clear effect on rumen passage kinetics (P ⩾ 0.081). Moreover, it showed that peak enrichments for ¹³C-DM and ¹³C-NDF in faeces were reached at 30.7 and 41.7 h post dosing, respectively. This is well after the time (24 h) when the ¹³C : ¹²C ratios of the in vitro unfermented residues have reached stable enrichment level. Fractional rate constants for particle passage from the rumen are estimated from the descending slope of faecal excretion curves. The present study shows that the decline in ¹³C : ¹²C ratio after peak enrichment is not affected by fermentative degradation and therefore can be used to assess feed component-specific fractional passage rates.     

The use of stable isotope ratios in whitebait otolith carbonate to identify the source of prey for Western Australian penguins

A methodology has been developed and used to investigate a direct link between juvenile whitebait (Hyperlophus vittatus), the key prey species sampled from the stomachs of Little Penguins (Eudyptula minor), and the specific nursery area of that prey. A unique application of existing methodology initially involved the measurement of the stable isotopes of oxygen (δ¹⁸O:δ¹⁶O) and carbon (δ¹³C:δ¹²C) in sagittal otolith carbonate, using standard mass spectrometric techniques. The results indicated that the inshore distribution of juvenile (0+ year old) whitebait between Cockburn Sound and Koombana Bay, Western Australia consisted of a number of separate assemblages. Measured differences in stable oxygen isotope ratios were attributed to variations in freshwater input to the embayments that provided the whitebait habitats. In contrast, the measured stable carbon isotope ratios probably resulted from the different isotopic compositions of the food webs in the various habitats. Secondly, a comparison of the average value of carbon and oxygen isotope signatures of pooled otoliths from samples of whitebait from a number of different nearshore coastal sites (assemblages), with that of whitebait obtained from the stomachs of penguins at their main breeding site (Penguin Island) indicated that the values from the penguins resemble most closely those of the average otolith values obtained from whitebait from only one site (Becher Point). Assuming that the whitebait sampled were representative of the whitebait in the nearshore habitats and the diets of the penguins, then these results imply that at the time of sampling the penguins were feeding on whitebait from only one site.     

Relationships between C3 Plant Foliar Carbon Isotope Composition and Element Contents of Grassland Species at High Altitudes on the Qinghai-Tibet Plateau,China

Relationships of foliar carbon isotope composition (δ¹³C) with foliar C, N, P, K, Ca, Mg contents and their ratios of 219 C₃ species leaf samples, obtained in August in 2004 to 2007 from 82 high altitude grassland sites on the Qinghai-Tibet Plateau China, were examined. This was done with reference to the proposition that foliar δ¹³C increases with altitude and separately for the life-form groups of graminoids, forbs and shrubs and for the genera Stipa and Kobresia. For all samples, foliar δ¹³C was negatively related to foliar K, P and ∑_{K+ Ca+ Mg}, and positively correlated to foliar C, C/N and C/P. The significance of these correlations differed for the taxonomic and life-form groups. Lack of a relationship of foliar δ¹³C with foliar N was inconsistent with the majority of studies that have shown foliar δ¹³C to be positively related to foliar N due to a decrease of C_i/C_a (the ratio between intercellular and atmospheric concentration of CO₂) and explained as a result of greater photosynthetic capacity at higher foliar N concentration. However this inconsistency relates to other high altitude studies that have found that photosynthetic capacity remains constant as foliar N increases. After accounting for the altitudinal relationship with foliar δ¹³C, of the elements only the K effect was significant and was most strongly expressed for Kobresia. It is concluded that factors critical to plant survival and growth at very high altitudes, such as low atmospheric pressure and low temperatures, may preclude expression of relationships between foliar δ¹³C and foliar elements that have been observed at lower altitudes.     

13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry

¹³C-based metabolic flux analysis (¹³CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured ¹³C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-¹³C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-¹³C] glucose. Decreasing the [1-¹³C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected ¹³C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.