The Application of 31P Nuclear Magnetic Resonance to Higher Plant Tissue: I. DETECTION OF SPECTRA

³¹P nuclear magnetic resonance (NMR) spectra are described fora variety of plant tissues, including sections of mature potatotuber (Solanum tuberosum) and sections of maize seedling (Zeamays). The experimental procedures for obtaining such spectra,using a conventional high field spectrometer, are discussedin detail. Assignments are given for the observed resonancesand the results are discussed in relation to the storage formsof plant phosphate. Attention is drawn to the power of the techniqueto distinguish the cytoplasm and vacuole in vivo, through thewell-known pH dependence of the ³¹P chemical shifts.     

Nuclear Magnetic Resonance of Tissue 23Na: II. Theoretical Line Shape

The theoretical line-shape function of the nuclear magnetic resonance (NMR) signal of ²³Na in biological tissue (and other unoriented systems) was obtained under the following conditions: (I) there occur two states of ²³Na in the system, (II) the exchange of ²³Na between the two states is rapid (but not too rapid), (III) in the absence of exchange, the ²³Na in one state is characterized by a single transverse relaxation time T₂ and a single Larmor frequency, and (IV) in the absence of exchange, the ²³Na in the other state possesses (a) two different values of T₂ and/or (b) more than one Larmor frequencies in the first order perturbation effect. The theoretical signal obtained consists of two Lorentzian components, which are centered at the same frequency, but characterized by different T₂. Only the narrower component, comprising 40% of the total intensity, is visible, when the fast T₂ is sufficiently short. The theoretical line-shape function of ²³Na signal was also calculated for oriented systems in which the above conditions are fulfilled.     

31P and 13C Nuclear Magnetic Resonance Studies of Macrophages

Biochemical events associated with differentiation and activation of monocyte-macrophage cell lines are of major interest in the understanding of pathophysiological processes as well as in research on immunopharmacological modulation of these cells. Nuclear magnetic resonance is the technique of choice for kinetic studies of metabolic events under such experimental conditions. This approach was used with the P388-D1 model of mature macrophages. Cells primed in vivo were triggered in vitro during NMR analysis and the results were compared to those from chemiluminescence tests performed simultaneously. Three preliminary phases were achieved: (i) ³¹P and ¹³C NMR spectroscopy of perchloric acid extracts, (ii) optimization of culture and perfusion conditions with validation of macrophage viability and functionality, and (iii) development of a data processing technique to improve the time resolution of kinetic studies. Based on their phosphocreatine content, cells primed in vivo exhibited greater maturation than control cells. After the respiratory burst of primed macrophages was triggered by concanavalin A, ³¹P NMR spectra reflected both a transient increase in ADP phosphorylation and intracellular acidification. ¹³C NMR studies indicated an acceleration of metabolism following in vitro triggering. The phenomenon was associated with an increased glucose consumption, implicating the hexose monophosphate shunt. These occurred concomitantly with the appearance of new peaks attributed to phosphorylated sugars.     

In Vivo 31P Nuclear Magnetic Resonance Investigation of Tellurite Toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by ³¹P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by ³¹P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

P-31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid-N₂-frozen guinea pig brains and their organophosphate profiles examined by P-31 nuclear magnetic resonance (NMR) spectroscopy. Thirty-two phosphorus-containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P-31 NMR is its pronounced ribose 5-phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide-induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α-glycerolphosphate and inorganic orthophosphate contents relative to control. P-31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P-31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5-phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3-phosphorylethanolamine (0.3δ); glycerol 3-phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri-(β-ATP) and di-(β-ADP) phosphate ionized end-groups (−6.2δ); α-ATP, α-ADP, and nicotinamide adenine dinucleotides esterified end-groups (−11.1δ); uridine diphosphohexose, hexose esterified end-groups (−13.0δ); and β-ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P-31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P-31 spectrum.     

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium

³¹P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn²⁺ and phosphate from the extracellular medium and on providing the O₂ supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose-6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied.     

P-31 Nuclear Magnetic Resonance Analysis of Brain: II. Effects of Oxygen Deprivation on Isolated Perfused and Nonperfused Rat Brain

Phosphatic metabolite (perchloric acid extractable) concentrations of cerebral tissues were analyzed by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy following external perfusion of the isolated rat brain (30 min or 60 min) under the following conditions: (a) constant perfusion pressure with either fluorocarbon- or erythrocyte-based medium, and (b) constant perfusate flow rate (3 ml/min) with the erythrocyte-based medium. Metabolite concentrations of control perfused brains were compared with those in nonperfused controls to provide a basis for detecting any qualitative or quantitative changes in cerebral metabolite composition. Metabolic responses of perfused brains to ischemia (incomplete ischemia, 83% reduction in flow for 10 min; transient complete ischemia for 1.5 or 2 min) were evaluated immediately after the ischemic episode and at selected time points during reperfusion (3 and 15 min). Alterations in cerebral metabolite levels induced by hypoxia were analyzed using a nonperfused rat brain model. Irrespective of the perfusion method employed, the phosphatic metabolites of control perfused rat brains were identical quantitatively to those of the nonperfused controls. Cerebral ischemia resulted in significantly increased levels of ADP, AMP + IMP, Pi, fructose 1,6-diphosphate, and glycerol 3-phosphate (global ischemia only), whereas ATP and phosphocreatine (PCr) levels declined significantly. The magnitude of these changes varied with the severity of the ischemia; however, following 15 min of control reperfusion metabolite levels had reverted to preischemic values. Significant perturbations in tissue phosphoethanolamine (3.84 delta resonance) content were evident at various time points during ischemia and postischemic recovery, which varied according to the perfusion conditions. In contrast to the changes observed in response to ischemia, hypoxia affected only cerebral high-energy phosphate levels. ATP and PCr levels were reduced, while a concomitant, essentially equimolar, increase in Pi and ADP was observed. The present studies indicate that in terms of phosphatic metabolites, the control equilibrated isolated perfused rat brain is quantitatively and qualitatively indistinguishable from the nonperfused rat brain in vivo regardless of the perfusion conditions (constant flow versus constant pressure). The metabolic responses to ischemia and hypoxia, as measured by P-31 NMR, were consistent with the pattern of changes reported elsewhere. Overall, P-31 NMR spectroscopic evaluation of the intact rat brain provides a potential experimental context for dynamic measures of cerebral metabolism under exogenously controlled conditions. Th     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Phytate Hydrolysis in Rat Gastrointestinal Tracts, as Observed by 31P Fourier Transform Nuclear Magnetic Resonance Spectroscopy

Phytate hydrolysis was followed through rat gastrointestinal tracts by ³¹P nuclear magnetic resonance spectroscopy. No phytate hydrolysis products were detected in the diet, stomach, or small intestine. It was concluded that cecal bacteria were responsible for phytate hydrolysis, which continued in the colon and fecal pellet.     

Analysis of Metabolism in Dormant Spores of Bacillus Species by 31P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

This work was undertaken to obtain information on levels of metabolism in dormant spores of Bacillus species incubated for weeks at physiological temperatures. Spores of Bacillus megaterium and Bacillus subtilis strains were harvested shortly after release from sporangia and incubated under various conditions, and dormant spore metabolism was monitored by ³¹P nuclear magnetic resonance (NMR) analysis of molecules including 3-phosphoglyceric acid (3PGA) and ribonucleotides. Incubation for up to 30 days at 4, 37, or 50°C in water, at 37 or 50°C in buffer to raise the spore core pH from ∼ 6.3 to 7.8, or at 4°C in spent sporulation medium caused no significant changes in ribonucleotide or 3PGA levels. Stage I germinated spores of Bacillus megaterium that had slightly increased core water content and a core pH of 7.8 also did not degrade 3PGA and accumulated no ribonucleotides, including ATP, during incubation for 8 days at 37°C in buffered saline. In contrast, spores incubated for up to 30 days at 37 or 50°C in spent sporulation medium degraded significant amounts of 3PGA and accumulated ribonucleotides, indicative of RNA degradation, and these processes were increased in B. megaterium spores with a core pH of ∼7.8. However, no ATP was accumulated in these spores. These data indicate that spores of Bacillus species stored in water or buffer at low or high temperatures exhibited minimal, if any, metabolism of endogenous compounds, even when the spore core pH was 7.8 and core water content was increased somewhat. However, there was some metabolism in spores stored in spent sporulation medium.     

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.     

Functional Imaging of Plants: A Nuclear Magnetic Resonance Study of a Cucumber Plant

Functional magnetic resonance imaging was used to study transients of biophysical parameters in a cucumber plant in response to environmental changes. Detailed flow imaging experiments showed the location of xylem and phloem in the stem and the response of the following flow characteristics to the imposed environmental changes: the total amount of water, the amount of stationary and flowing water, the linear velocity of the flowing water, and the volume flow. The total measured volume flow through the plant stem was in good agreement with the independently measured water uptake by the roots. A separate analysis of the flow characteristics for two vascular bundles revealed that changes in volume flow of the xylem sap were accounted for by a change in linear-flow velocities in the xylem vessels. Multiple-spin echo experiments revealed two water fractions for different tissues in the plant stem; the spin-spin relaxation time of the larger fraction of parenchyma tissue in the center of the stem and the vascular tissue was down by 17% in the period after cooling the roots of the plant. This could point to an increased water permeability of the tonoplast membrane of the observed cells in this period of quick recovery from severe water loss.     

核磁共振技术在蛋白质领域的应用

简要叙述了核磁共振技术(NMR)在蛋白质领域的研究及应用。NMR法通过测定蛋白质在稀溶液状态下反应位点的特定参数来计算蛋白质的三级结构,并可深入了解一定时间范围内化学反应和蛋白质构象转变的动力学过程。通过NMR对抗原决定簇和抗体CDR作图,可以分析其一级结构和三维构象;对抗原抗体动力学的分析,对于设计基因疫苗、检测细胞表面抗原提呈以及分析抗原抗体复合物的构象变化也有着重要意义。     

Effect of External pH on the Cytoplasmic and Vacuolar pHs in Mung Bean Root-Tip Cells: A 31P Nuclear Magnetic Resonance Study

The effect of the external pH on the intracellular pH in mungbean (Vigna mungo (L.) Hepper) root-tip cells was investigatedwith the ³¹P nuclear magnetic resonance (NMR) method. The ³¹PNMR spectra showed three peaks caused by cytoplasmic G-6-P,cytoplasmic P_i and vacuolar P_i. The cytoplasmic and vacuolarpHs could be determined by comparing the P_i chemical shiftswith the titration curve. When the external pH was changed overa range from pH 3 to 10, the cytoplasmic pH showed smaller changesthan the vacuolar pH, suggesting that the former is regulatedmore strictly than the latter. The H⁺-ATPase inhibitor, DCCD,caused the breakdown of the mechanism that regulates the intracellularpH. H⁺-ATPase appears to have an important part in the regulationof the intracellular pH. (Received January 4, 1984; Accepted August 27, 1984)     

Nuclear Magnetic Resonance of Tissue 23Na: I. 23Na Signal and Na+ Activity in Homogenate

The ability to depress the resonance intensity of ²³Na in rat liver tissue was not found in the supernatant fraction. It was exclusively localized in particulate fractions. The intensity and saturation behavior of the ²³Na signal was examined in suspensions containing various amounts of the particulate fraction of rat liver homogenate. The results strongly suggest that the ²³Na signal of tissue reflects quadrupole interactions and does not result from a slow exchange between the free and bound fractions of Na⁺. The activity coefficient of Na⁺ in rat liver homogenate (no medium was added) was 0.59, about 20% less than that in the isotonic saline. Available evidences and discussion indicate that the bound Na⁺ in the homogenate is much less than the so-called “NMR-invisible” fraction of Na⁺.     

Lymphocyte Activation-31P Magnetic Resonance Studies of Energy Metabolism and Phospholipid Pathways

³¹P NMR spectra of perfused lymphocytes embedded in alginate capsules and activated by interleukin-2 (IL-2) are remarkably different from those of control lymphocytes. The main differences are the appearance and gradual increase of phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occur following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and are not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibits the effects of PHA, but not of IL-2. There are no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine (PC) and phosphatidylethanolamine is an inherent feature of the activation process. ³¹P NMR spectra of lymphocytes are characterized by a low phosphocholine signal. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of phosphocholine cytidyltransferase, indicate that choline kinase plays a key role in regulating PC synthesis in human lymphocytes.     

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。     

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.     

Phase-Locked Loop Technique to Record Resonance Frequency of Plant Tissue

An apparatus based on the phase-locked loop technique has been developed in order to record automatically the resonance frequency of a mechanically vibrating plant specimen. Hereby changes in the elasticity of the plant material can be continuously recorded. In order to demonstrate the use of the apparatus, elasticity changes of Avena coleoptiles due to exchange of the root medium were continuously recorded.