Bulk analysis of tobacco and cigarettes by magnetic resonance imaging.

Proton magnetic resonance imaging of tobacco blends in cigarette rods was investigated to assess the feasibility of various imaging protocols to characterize and quantify the structure and composition of multiphase plant materials in situ. The protocols used to characterize the rigid molecular components (plant cell wall) included single-point imaging (SPI) and a variant experiment, single-point ramped imaging with T(1) enhancement (SPRITE). Both 1D profiles, radially averaged along the length of a cigarette, and 2D maps of proton spin density and relaxation (T(2)) were acquired. Mobile components (tobacco waxes and water) were examined via conventional spin-echo imaging techniques, with 1D, 2D, and 3D data being acquired. Spin-spin relaxation times (T(2)), apparent spin-spin relaxation (T(2)), and spin-lattice (T(1)) relaxation times were measured for selected samples.

Lignification in cell suspension cultures of Pinus taeda. In situ characterization of a gymnosperm lignin.

Pinus taeda suspension cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed only primary cell wall formation and essentially no lignification, as determined by histochemical, ultrastructural, chemical, and NMR spectroscopic analyses. However, these cultures maintained a functional phenylpropanoid pathway as demonstrated by formation of the lignans (-)-matairesinol and (-)-pinoresinol. Administration of [1-13C]Phe to these cultures, followed by solid-state carbon-13 NMR spectral analysis of their cell walls, demonstrated that the phenylalanine incorporated into the cell wall matrix was primarily as protein, rather than lignin. Successive transfer of the 2,4-dichlorophenoxyacetic acid-grown cultures to alpha-naphthaleneacetic acid-containing medium induced cell wall thickening concomitant with lignification. The presence of lignin was confirmed by histochemical, ultrastructural, chemical, biochemical, and NMR spectroscopic analyses. Specific labeling of the lignin polymer in situ with [1-13C]-, [2-13C]-, and [3-13C]Phe and analysis of the cell wall preparations by solid-state carbon-13 NMR spectroscopy permitted the first direct determination of the in situ bonding patterns in a gymnosperm lignin. Several dominant interunit linkages were observed, including beta-O-aryl, furanofuran, phenylcoumarin, and phenolic-linked monolignols, consistent with those predicted but hitherto not proven. Finally, milled wood lignin derivatives prepared from these 13C-specifically enriched lignin tissues gave a relatively high fidelity copy of the native lignin.

Monitoring biosynthesis of wheat cell-wall phenylpropanoids in situ.

Lignins and suberins are complex plant cell-wall macromolecules that are composed mainly of phenylpropanoid residues derived from L-phenylalanine. Lignins and suberins are considered to be covalently linked to carbohydrates and to lipids, respectively. The bonding of these important structural materials within cell walls has never been established. By feeding specifically labeled [(13)C] ferulic acid over extended durations to seedlings of Triticum aestivum L. and by using solid-state carbon-13 nuclear magnetic resonance techniques, the major resonances due to specific carbons in the propanoid side chains of these cell-wall polymers have been identified in situ. The signals were found to differ significantly from those of synthetic lignins, which have usually been considered to be good approximations of natural lignin structure.

Characterization of alternating deoxyribonucleic acid conformations in solution by phosphorus-31 nuclear magnetic resonance spectroscopy.

Medium length (500-200 bp) alternating purine-pyrimidine DNAs were prepared by sonication of synthetic polymers at low temperature. The products, and the hairpin structures derived from them after melting, were sufficiently small for high-resolution 31P NMR studies. Of the five sequences studied, two DNAs, poly(dG-dC).poly(dG-dC) and poly(dA-dU).poly(dA-dU), gave singlet 31P resonances, while three others, poly(dA-dT).poly(dA-dT), poly(dA-br5U).poly(dA-br5U), and poly(dI-dC).poly(dI-dC), exhibited two resolved signals of equal area. This indicates the existence of two distinct alternating phosphodiester backbone conformations for these latter three B-DNAs in solution. Controls of homopolymers, which were also prepared by sonication, showed only singlet 31P resonances. Of the alternating sequences DNAs, only sonicated poly(dG-dC).poly(dG-dC) exhibited a conformational transition to a high salt (greater than 2.5 M) form which exhibited two well-resolved 31P resonances of equal area. This indicates that the high salt form of poly(dG-dC).poly(dG-dC) also has an alternating backbone structure, and it is presumed to be a Z-DNA. These results indicate a general response of the DNA backbone conformation to alternating purine-pyrimidine base sequences but with a degree of sequence and environmental specificity which might have functional genetic significance.

Geometry of the phosphodiester backbone in the A form of deoxyribonucleic acid determined by phosphorus-31 nuclear magnetic resonance spectroscopy.

31P NMR spectra were obtained from poly(dA-dT) . poly(dA-dT) fibers which gave an X-ray diffraction pattern similar to that of the A form of natural deoxyribonucleic acid (DNA). The analysis of the line shape indicated that the A form of poly(dA-dT) . poly(dA-dT) has a single uniform backbone conformation; the orientation of the phosphodiester group relative to the helical axis was determined to be beta = 70 degrees and gamma = 52 degrees. The 31P NMR spectra of poly(dA-dT) . poly(dA-dT) were in remarkable contrast to the 31P NMR spectra of the A form of natural DNA, which exhibited an unusual line shape. The origins of the abnormalities in the line shape for the A form of natural DNA are discussed in terms of phosphodiester orientations.

Nonuniform backbone conformation of deoxyribonucleic acid indicated by phosphorus-31 nuclear magnetic resonance chemical shift anisotropy.

31P nuclear magnetic resonance of highly oriented DNA fibers has been observed for three different conformations, namely, the A, B, and C forms of DNA. At a parallel orientation of the fiber axis with respect to the magnetic field, DNA fibers in both the A and B forms exhibit a single, abnormally broad resonance; in contrast, fibers in the C form show almost the full span of the chemical shift anisotropy (170 ppm). The spectra of the fibers oriented perpendicular indicate that the DNA molecules undergo a considerable rotational motion about the helical axis, with a rate of greater than 2 x 10(3) s-1 for the B-form DNA. Theoretical considerations indicate that the 31P chemical shift data for the B-form DNA fibers are consistent with the atomic coordinates of the phosphodiester group proposed by Langridge et al. [Langridge, R., Wilson, H. R. Hooper, C. W., Wilkins, M. H. F., & Hamilton, L. D. (1960) J. Mol. Biol. 2, 19--37] but not with the corresponding coordinates proposed by Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Coomun. 47, 1504--1509], and also lead to the conclusion that the phosphodiester orientation must vary significantly along the DNA molecule. The latter result suggests that DNA has significant variations in its backbone conformation along the molecule.

Protein mobility and self-association by deuterium nuclear magnetic resonance.

Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.