Spinning-sideband-free and spinning-sideband-only NMR spectra in spinning samples

Dixon, William J.

doi:10.1063/1.444076

Cited by 593 publications

(345 citation statements)

References 14 publications

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“…4, upper left), since the total phosphorus in the added KH2PO4 is approximately 5 times that normally found in alfalfa callus (cf below).…”

Section: Resultsmentioning

confidence: 99%

Regeneration in Alfalfa Tissue Culture

Schaefer

1985

Plant Physiol.

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ABSTRACrThe composition of phosphos-containing compouds of intact lyophilized alfalfa tissue has been determned, in part, by soLid-state 31p nuclear magnetic resonance. The tissue (Mcdicago sativa L, cv Regen S; and some of its crosses) was grown in culture under both nonregeneating and regenerating conditions, the latter enhnced by the addition of specific amino acids. Analysis of the 31P nouclear m etic resonance spectra shows that regeneration is favored when metabolism occurs without the production of a low average intracelular pH.Phosphorus-31 NMR is widely used in biological studies of intact cells. The levels of ATP, phosphocreatin, and Pi are often diagnostic of the metabolic state of mammalian tissue (6), and have been used in both research and clinical applications in studying, for example, the effect of pH in determining cardiac function (7), the biochemical basis for muscle fatigue (4), and the protection of organs for transplants during prolonged ischemia (1). Plant tissues can be examined by 31P NMR as well. Recently, the intracellular pH of maize root tips has been measured by 31p NMR (12), and related to cytoplasmic acidosis resulting from growth under anaerobic conditions (1 1, 13 chemical information in the spectra. Cross-polarization of31P by abundant 'H makes the high-resolution solid-state experiment a high-sensitivity one as well (15). CPMAS 31P NMR has been used before in biological systems to examine the composition of bone (2, 14) and for the characterization of metabolism in developing amphibian embryos (3). In this paper, we present evidence from solid-state 31P NMR that the extent of regeneration in alfalfa cell culture is influenced by the intracellular pH. MATERIALS AND METHODSPlant Material and Culture Procedures. The three alfalfa genotypes used in these experiments, their method of culture, and the preparation of intact tissue for NMR analysis are described in detail in the preceding paper (17). 3lP NMR. CPMAS 31P NMR spectra were obtained at 36.2 MHz using matched spin-lock cross-polarization transfers with 1-ms single contacts and 35-kHz radio-fiequency fields with the lyophilized intact cell samples contained in a Beams-Andrew 420 ,ul hollow rotor spinning at 1.2 kHz. Technical details of the spinning and cross-polarization procedures may be found elsewhere (16). Spectra are presented on ppm chemical shift scale referenced to external solid phosphocreatin (0 ppm).Total phosphorus was deermined by integration of CPMAS 31p NMR spectra calibrated by a standard of lkown 31P content.Potassium concentration was determined by elemental analysis. Total cellular calcium and sodium levels of a few samples were determined by argon plasma optical emission spectroscopy. Standard pH determinations were made using a Horizon model 5998-10 pH meter calibrated with a Fisher certified buffer solution.Extraction Procedure. The ethanol-water insoluble fraction was isolated by grinding intact tissue in a mortar under an 80% ethanol 20% water mixture. This material was then spun at l0,OOOg for a few...

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“…4, upper left), since the total phosphorus in the added KH2PO4 is approximately 5 times that normally found in alfalfa callus (cf below).…”

Section: Resultsmentioning

confidence: 99%

Regeneration in Alfalfa Tissue Culture

Schaefer

1985

Plant Physiol.

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“…Most of the 13 C spectra in Fig. 1 were recorded under magic-angle spinning of 5.3 kHz with total suppression of sidebands (38). The cross polarization contact time was 1 ms and the recycle delay 2.5 s. To select signals of carbons with weak C─H dipolar couplings, spectra were also acquired with additional 40 μs of gated decoupling before signal detection.…”

Section: Methodsmentioning

confidence: 99%

Strongly bound citrate stabilizes the apatite nanocrystals in bone

Rawal

Schmidt‐Rohr

2010

Proc. Natl. Acad. Sci. U.S.A.

451

494

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Nanocrystals of apatitic calcium phosphate impart the organicinorganic nanocomposite in bone with favorable mechanical properties. So far, the factors preventing crystal growth beyond the favorable thickness of ca. 3 nm have not been identified. Here we show that the apatite surfaces are studded with strongly bound citrate molecules, whose signals have been identified unambiguously by multinuclear magnetic resonance (NMR) analysis. NMR reveals that bound citrate accounts for 5.5 wt% of the organic matter in bone and covers apatite at a density of about 1 molecule per ð2 nmÞ 2 , with its three carboxylate groups at distances of 0.3 to 0.45 nm from the apatite surface. Bound citrate is highly conserved, being found in fish, avian, and mammalian bone, which indicates its critical role in interfering with crystal thickening and stabilizing the apatite nanocrystals in bone.T he load-bearing material in bone is a fascinating organicinorganic nanocomposite whose stiffness is provided by thin nanocrystals of carbonated apatite, a calcium phosphate, imbedded in an organic matrix consisting mostly of collagen, a fibrous protein (1-5). The small (ca. 3-nm) thickness of the apatite nanocrystals is favorable for mechanical properties, likely preventing crack propagation (6). While the size and shape of the nanocrystals have been studied extensively (4, 5), the mechanism stabilizing them at a thickness corresponding to only about four unit cells has not been elucidated. A better understanding of the factors controlling the nanocrystals in bone is desirable for prevention and treatment of bone diseases such as osteoporosis, which causes millions of fractures each year (7), and for more efficient synthesis of biomimetic nanocomposites (8, 9). In vitro experiments have shown that carboxylate-rich proteins such as osteocalcin and osteopontin (7) can affect hydroxyapatite crystal formation and growth (10, 11). These observations might suggest that such proteins limit nanocrystal thickening (12); however, these proteins are not sufficiently abundant in vivo to bind to all the nanocrystal surfaces at high enough area concentration; possibly, they control the length of the nanocrystals (7).Here we show instead that the surfaces of the apatite crystals in bone are studded with strongly bound citrate molecules, at a density of ca. 1∕ð2 nmÞ 2 , using advanced solid-state nuclear magnetic resonance (NMR) as a unique tool for probing buried interfaces. Citrate is quite abundant in bone (ca. 1 wt%, or 5 wt% of the organic components) (13,14). Before 1975, citrate in bone was studied by simple wet-chemical methods and thought to regulate bone demineralization (14). However, citrate is no longer even mentioned in most of the prominent literature on the bone nanocomposite published during the last thirty years (1)(2)(3)(4)(5)(15)(16)(17)(18). We now highlight the importance of citrate in bone by demonstrating that it is not a dissolved calcium-solubilizing agent but a strongly bound, integral part of the nanocomposite. Structurally, citrate s...

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“…Twenty-four t1 increments with a dwell time of 10 µs were used in the 2D experiments. Before detection, the TOSS fourpulse sequence 17 was applied to suppress spinning side bands.…”

Section: Wise-2d and Spin Diffusion Experimentsmentioning

confidence: 99%

Late Stages of Phase Separation in a Binary Polymer Blend Studied by Rheology, Optical and Electron Microscopy, and Solid State NMR

et al. 1997

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The relation between rheology and the time dependent morphology of a phase-separating binary blend of polystyrene and poly(vinyl methyl ether) was investigated by heating a sample from the single-phase (at 90°C) into the two-phase regime (at 124°C, 16 K above the LCST) and maintaining its temperature there while measuring the evolution of the dynamic moduli G′ and G′′. Morphological changes occurred slowly so that there was sufficient time to cycle the dynamic mechanical measurements repeatedly over five decades in frequency. The morphology was observed on length scales from 1 mm down to 1 nm by conventional optical microscopy combined with digital image analysis, Hoffman modulation microscopy, TEM, and WISE NMR with spin diffusion. NMR shows that major compositional changes occur mostly in the first 20 min and then the composition remains constant at about 60:40 PS/PVME for the PS-rich matrix and 5:95 PS/PVME for the PVME-rich microdomains. The PVME-rich microdomains are separated by thin layers of the PS-rich phase which forms the matrix. On a larger scale, shape and geometry change during the entire experiment (42 h). The linear domain growth appears to be consistent with the theories of Siggia and Doi-Ohta. The initial increase of the dynamic moduli is attributed to the formation of highly interconnected PVME-rich and PS-rich phases during spinodal decomposition. The subsequent decrease of the values of the dynamic moduli is considered to be the result of the loss of the interconnectivity between the two phases due to the breakup of the PS-rich phase network and the coalescence of the PVME-rich domains.

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Spinning-sideband-free and spinning-sideband-only NMR spectra in spinning samples

Cited by 593 publications

References 14 publications

Regeneration in Alfalfa Tissue Culture

Regeneration in Alfalfa Tissue Culture

Strongly bound citrate stabilizes the apatite nanocrystals in bone

Late Stages of Phase Separation in a Binary Polymer Blend Studied by Rheology, Optical and Electron Microscopy, and Solid State NMR

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