Characterisation of Soybean and Wheat Seeds by Nuclear Magnetic Resonance Spectroscopy

The effects of equilibration under different air relative humidities (RH, 1 – 90 %) and temperatures (35 and 45 °C) on soybean (Glycine max) and wheat (Triticum aestivum) seeds were studied using different techniques. Seed moisture content, electrical conductivity (EC) of seed leachate and per cent seed germination were measured following standard procedures, and compared with nuclear magnetic resonance spin-spin relaxation time (T₂) measurements. Moisture contents of soybean and wheat seeds, following the reverse sigmoidal trend, were greater at 35 than at 45 °C at any particular RH. Changes in T₂ were related to the changes in germination percentage and leachate EC of both soybean and wheat seeds. Equilibrating soybean seeds at RH 11 % decreased germination percentage with corresponding decrease in T₂. On the contrary, EC of seed leachate increased. In wheat seeds equilibrated at 45 °C, T² was maximal at RH 5.5 %. T₂ declined in seeds equilibrated at high RH (> 80 %) together with low germination percentage.     

Classification of Modern Resins by Solid State Nuclear Magnetic Resonance Spectroscopy

The carbon-13 nuclear magnetic resonance (NMR) spectra of modern resins have been used to characterize their botanical sources. Resins from four of the principal diterpenoid resinproducing genera, Agathis, Hymenaea, Pinus, and Araucaria, give distinct NMR spectra under conditions of normal and interrupted decoupling. The genus Wollemia gives the same spectra as Agathis. Samples from the triterpenoid resin-producing family Burseraceae and of gum resins were recorded for comparison. Resin samples from unknown or uncertain sources then were examined and assigned to their botanical sources.     

Estimation of Ammonium Ion Distribution between Cytoplasm and Vacuole Using Nuclear Magnetic Resonance Spectroscopy

Evidence is presented that intracellular ammonium is trapped in vacuoles of maize (Zea mays L.) root tips because of rapid movement of ammonia between cytoplasm and vacuoles. The concentration of cytoplasmic ammonium is estimated to be <15 μm at extracellular ammonium concentrations up to 1 mm. The implications for pathways of ammonium assimilation are discussed.     

生物大分子质谱电离技术的突破及核磁共振三维结构测定方法的建立——2002年诺贝尔化学奖简介

2002年诺贝尔化学奖授予了质谱和核磁共振领域的三位科学家以表彰他们对生物大分子鉴定及结构分析方法做出的贡献．其中两位科学家J．B．Fenn和K．Tanaka分别发展了生物大分子质谱分析的软解吸电离方法;另一科学家K．Wüthrich则将核磁共振技术成功地应用于生物大分子如蛋白质的溶液三维结构测定．他们的研究成果已使质谱和核磁共振技术成为生物大分子强有力的研究手段,极大地促进了生物大分子的研究进程,必将对整个生命科学研究产生深远的影响．     

Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo³¹P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH ~6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N₂, the cytoplasmic Pi increased ~150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of ~10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H⁺-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.     

Flocculation Effects on Bound Water in Sludges as Measured by Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance relaxation times (T₁ and T₂) were measured for flocculated and unflocculated samples of activated sludge. The weight of water and solids in the sludge samples was found and related to T₁ to find the relative percentage of bound water. The results suggest that the amount of bound water increases as the samples become more unflocculated. The values of T₁ and T₂ also indicate that unflocculated individual particles are characterized by loose packing of shorter molecules and that the addition of larger molecules may induce flocculation.     

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The ¹³C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional ¹⁵N and ³¹P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.     

Interaction of Amino Acids with Transition Metal Ions in Solution (I) Solution Structure of l-Lysine with Co(II) and Cu(II) Ions as Studied by Nuclear Magnetic Resonance Spectroscopy

Interaction of l-lysine with Co(II) and Cu(II) ions has been studied using ¹H- and ¹³C-NMR and solution absorption spectrometry. In l-lysine-Co²⁺ solution in D₂O (100: 1 in concentration), coordination interaction of the α-amino and carboxyl groups with Co²⁺ occurs from the neutral to alkaline pD region, whereas no interaction of the ?-amino group was observed throughout the whole pD region. On the other hand, in l-lysine-Cu²⁺ solution, the ?-amino group also takes part in complexation in the higher pD region (pD≧10). Structural changes in complexation of l-lysine with the divalent cations along with pD variations in aqueous solution are discussed. Dissociation constants of the three functional groups were obtained by ¹H-NMR chemical shifts; pKa₁ = 2.2, pKa₂ = 9.5 and pKa₃ = 11.2.     

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.     

Characterization of Metabolites in IntactStreptomyces citricolorCulture Supernatants Using High-Resolution Nuclear Magnetic Resonance and Directly Coupled High-Pressure Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional 1H NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures of Streptomyces citricolor. By applying 1H NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC-NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the 1H NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as 1H-1H correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on 1H-1H coupling networks and 13C chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment.     

Fungal Metabolism of Toluene: Monitoring of Fluorinated Analogs by 19F Nuclear Magnetic Resonance Spectroscopy

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by ¹⁹F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the ¹⁹F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.     

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering,Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.     

Ethanol Reassimilation and Ethanol Tolerance in Pichia stipitis CBS 6054 as Studied by C Nuclear Magnetic Resonance Spectroscopy

Ethanol reassimilation in Pichia stipitis CBS 6054 was studied by using continuous cultures, and the oxidation of [1-C]ethanol was monitored by in vivo and in vitro C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in P. stipitis compared with that in Saccharomyces cerevisiae (S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).     

Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.     

Carbon-13 Nuclear Magnetic Resonance Analysis of Metabolism in Soybean Labeled by CO(2)

Fourier transform ¹³C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules (Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to ¹³CO₂ 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway.     

Evaluation of Metabolomic Changes as a Biomarker of Chondrogenic Differentiation in 3D-cultured Human Mesenchymal Stem Cells Using Proton (1H) Nuclear Magnetic Resonance Spectroscopy

Purpose

The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using ¹H nuclear magnetic resonance (NMR) spectroscopy.

Materials and Methods

hMSCs (2×10⁶ cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (600-MHz) NMR with the water suppression sequence, zgpr. Real-Time polymerase chain reaction (PCR) was performed to confirm that that the hMSCs differentiated into chondrocytes and to analyze the metabolomic changes indicated by the NMR spectra.

Results

During chondrogenesis, changes were detected in several metabolomes as hMSC chondrogenesis biomarkers, e.g., fatty acids, alanine, glutamate, and phosphocholine. The metabolomic changes were compared with the Real-Time PCR results, and significant differences were determined using statistical analysis. We found that changes in metabolomes were closely related to biological reactions that occurred during the chondrogenesis of hMSCs.

Conclusions

In this study, we confirm that metabolomic changes detected by ¹H-NMR spectroscopy during chondrogenic differentiation of 3D-cultured hMSCs in alginate beads can be considered as biomarkers of stem cell differentiation.     

Carbon Metabolism in Spores of the Arbuscular Mycorrhizal Fungus Glomus intraradices as Revealed by Nuclear Magnetic Resonance Spectroscopy

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO(2) is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state.     

Intracellular pH of Cotton Embryos and Seed Coats during Fruit Development Determined by P Nuclear Magnetic Resonance Spectroscopy

The pH of the phosphate-containing compartments of developing cotton seed coat and embryo tissues was determined by means of ³¹P nuclear magnetic resonance spectroscopy. The pH values of these tissues varied as a function of developmental age. From 27 to approximately 38 days postanthesis, a strong pH differential existed between the two tissues; the seed coat was up to 1.4 pH units more acid than developing cotton embryos. The pattern of pH values found with this technique agrees with pH values of tissue homogenates in distilled water. The results confirm an earlier suggestion that seed coat cells are more acidic than embryo cells during key developmental stages of the seed. The pH differential between these two tissues causes abscisic acid to diffuse from seed coats to embryos against its apparent concentration gradient to prevent viviparous germination, despite a higher abscisic acid concentration in the embryo.     

Ethanol Reassimilation and Ethanol Tolerance in Pichia stipitis CBS 6054 as Studied by 13C Nuclear Magnetic Resonance Spectroscopy

Ethanol reassimilation in Pichia stipitis CBS 6054 was studied by using continuous cultures, and the oxidation of [1-¹³C]ethanol was monitored by in vivo and in vitro ¹³C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in P. stipitis compared with that in Saccharomyces cerevisiae (S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).