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.     

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.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

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.     

Proton Nuclear Magnetic Resonance Spectroscopy of Rabbit Brain Homogenate

Abstract: Proton nuclear magnetic resonance (¹H NMR) spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.     

Quantitative Determination of Site-Specific Conformational Distributions in an Unfolded Protein by Solid-State Nuclear Magnetic Resonance

Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state ¹³C NMR spectra of samples prepared with uniform ¹³C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone ? and ψ torsion angles in samples with sequential pairs of carbonyl ¹³C labels. Without GdnHCl, the combined data are well fit by α-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of α-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.     

核磁共振波谱法在蛋白质三维结构解析中的应用

蛋白质特定的三维结构与其生物功能密切相关,因此,研究蛋白质的三维结构有助于揭示其生物功能机制。将核磁共振（NMR）波谱法应用于研究溶液状态下蛋白质的三维结构,能够更加准确地揭示蛋白质结构与生物功能之间的关系。本文综述了NMR解析蛋白质三维结构的理论和技术方法,以及NMR结合其他生物物理手段,并辅以分子建模计算法研究蛋白质三维结构的研究进展和最新方法,为精准解析蛋白质的三维结构提供思路及策略。     

Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods

The time course of suberization in wound periderm from potato (Solanum tuberosum L.) has been monitored by histochemical and high-resolution solid-state nuclear magnetic resonance (NMR) methods. Light microscopy conducted after selective staining of the lipid and double-bonded constituents shows that suberin is deposited at the outermost intact cell-wall surface during the first 7 d of wound healing; suberization forms a barrier to tissue infiltration at later times. Cross polarization-magic angle spinning 13C NMR spectra demonstrate the deposition of a polyester containing all major suberin functional groups after just 4 d of wound healing. Initially the suberin includes a large proportion of aromatic groups and fairly short aliphatic chains, but the spectral data demonstrate the growing dominance of long-chain species during the period 7 to 14 d after wounding. The results of preliminary 13C-labeling experiments with sodium [2-13C]acetate and DL-[1-13C]phenylalanine provide an excellent prospectus for future NMR-based studies of suberin biosynthesis.     

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.     

Proton Nuclear Magnetic Resonance Spectroscopy of Primary Cells Derived from Nervous Tissue

Abstract: Cell culture techniques, high-resolution in vitro ¹H nuclear magnetic resonance (NMR) spectroscopy, and chromatographic analyses were used to compare the properties of purified cell populations derived from the PNS and cortical neurones. Cell cultures were immunocytochemically characterised with specific antibodies to ensure purity of the individual cultures. Spectra of perchloric acid extracts of cultured Schwann cells, perineural fibroblasts, dorsal root ganglion neurones, and cortical neurones displayed several common features. However, statistically significant differences were found by ¹H NMR spectroscopy in most metabolites among the cell types studied. In addition, cells could be distinguished by the presence or absence of certain amino acids. For example, N -acetylaspartate was present in dorsal root ganglion neurones and cortical neurones, γ-aminobutyric acid was present in large amounts in cortical neurones, and Schwann cell spectra displayed a large signal from glycine. These results extend our earlier findings that different cell types of the CNS exhibit highly characteristic metabolite profiles to now include the major cell types of the PNS. These latter cell types also exhibit characteristic metabolite compositions, such that even Schwann cells and oligodendrocyte type 2 astrocyte (O-2A) progenitor cells—precursors of the myelinating cells of the CNS and PNS, respectively—can be readily distinguished from each other.     

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with ¹³C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of ¹³C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.     

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.     

Solid-State 13C Nuclear Magnetic Resonance Characterization of Cellulose in the Cell Walls of Arabidopsis thaliana Leaves

Solid-state 13C nuclear magnetic resonance was used to characterize the molecular ordering of cellulose in a cell-wall preparation containing mostly primary walls obtained from the leaves of Arabidopsis thaliana. Proton and 13C spin relaxation time constants showed that the cellulose was in a crystalline rather than a paracrystalline state or amorphous state. Cellulose chains were distributed between the interiors (40%) and surfaces (60%) of crystallites, which is consistent with crystallite cross-sectional dimensions of about 3 nm. Digital resolution enhancement revealed signals indicative of triclinic and monoclinic crystalline forms of cellulose mixed in similar proportions. Of the five nuclear spin relaxation processes used, proton rotating-frame relaxation provided the clearest distinction between cellulose and other cell-wall components for purposes of editing solid-state 13C nuclear magnetic resonance spectra.     

Degradation of 4-Fluorobiphenyl by Mycorrhizal Fungi as Determined by 19F Nuclear Magnetic Resonance Spectroscopy and 14C Radiolabelling Analysis

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using ¹⁹F nuclear magnetic resonance (NMR) spectroscopy in combination with ¹⁴C radioisotope-detected high-performance liquid chromatography (¹⁴C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. ¹⁴C-HPLC profiles indicated that there were four major biotransformation products, whereas ¹⁹F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4′-ol and 4-fluorobiphen-3′-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.     

Fingerprinting Analysis of Saposhnikovia divaricata using 1H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography

The 1H nuclear magnetic resonance (1H NMR) fingerprints of fractionated non-polar and polar extracts (control substance for plant drug [CSPD] A and B) from the roots of 12 specimens of Saposhnikovia divaricata (Turcz.) Schischk were achieved with Fourier Transform (FT)-NMR spectrometer and assigned by comparison to each other and to the 1H NMR spectra of the isolated individual compounds. These fingerprints were found to be uniform in terms of the specificity for the implication of all 12 specimens being systematically of the same origin. The uniformity was further affirmed by high performance liquid chromatography (HPLC), which also revealed exactly identical specificity for the identified S. divaricata species with the 1H NMR appearances of corresponding CSPD on the part of the composition of characteristic constituents when comparing to corresponding individual compounds. This investigation unambiguously shows that the specific signals from the chemotaxonomically significant compounds of chromones and coumarins in S. divaricata are exhibited distinctively in the composite features of both 1H NMR fingerprints and HPLC profiles. The 1H NMR and HPLC profiles established can successfully be used as reference for the authentication of the origin of S. divaricata species as well as for chemotaxonomic studies.     

13C Solid-State Cross Polarization/Magic-Angle-Spinning Nuclear Magnetic Resonance Spectra of Natural and Synthetic Melanins

The availability of NMR spectrometers operating in cross polarization/magic angle spinning (CP-MAS) has provided a powerful tool for the structural elucidation of insoluble materials. In this ¹³C NMR study of eumelanins we report the first direct evidence of the presence of different chemical functionalities in synthetic and natural eumelanins. These spectra contain useful information for the characterization of melanins from different sources.     

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.     

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.