Drug release from cast films of ethylene vinyl acetate (EVA) copolymer: Stability of drugs by 1H NMR and solid state 13C CP/MAS NMR.

The study utilizes an oral biocompatible material based on ethylene vinyl acetate copolymer (EVA) designed to release drugs in vitro at therapeutic levels over several days. We examined the drug stability during film casting process using proton and solid state NMR techniques. The drug-loaded EVA films were prepared from the dry sheet obtained by solvent (dichloromethane) evaporation of polymer casting solutions. Drugs tested include chlorhexidine diacetate (CDA), doxycycline hydrochloride (DOH), tetracycline hydrochloride (TTH) and nystatin (NST). Drug release from the films was examined for at least 14 days in 10 ml ddH2O (NST in water/ethanol (4:1)) which was replaced daily. Changes in optical density were followed spectraphotometrically. Effect of temperature on rate measurements was studied and the energies of activation (E*) were calculated using Arrhenius plots. Effect of EVA copolymer composition on CDA release rate was also investigated. The enhanced rates with temperature increase may be attributed to the formation of channels with increased geometry in the polymer. The highest E* observed for CDA compared to DOH and TTH may be related to their average molecular weights. Spectral analyses for CDA and NST revealed that the chemical and physical structures of the drugs remained unaffected during the film casting process.

Analysis of a dimethacrylate copolymer (bis-GMA and TEGDMA) network by DSC and 13C solution and solid-state NMR spectroscopy.

In this work the effect of dilution with TEGDMA on the kinetics of Bis-GMA polymerization and on the extent of polymerization or degree of conversion was studied using (a) DSC and (b) NMR. The systems with lower viscosity and lower Tg exhibited higher extent of polymerization. For Bis-GMA/TEGDMA mixtures the calculated Tg values were found to be higher than the experimental values suggesting that a dilution effect is predominant rather than intermolecular hydrogen bonding. Solid state NMR has been shown to be a convenient method for measuring the total amount of conversion in a mixed monomer system. The disappearance of the NMR solution spectrum was used to reveal overall polymerization kinetics.

Protein dynamics from chemical shift and dipolar rotational spin-echo 15N NMR.

The partial collapse of dipolar and chemical shift tensors for peptide NH and for the amide NH at cross-link sites in cell wall peptidoglycan, of intact lyophilized cells of Aerococcus viridans, indicates NH vector root-mean-square fluctuations of 23 degrees. This result is consistent with the local mobility calculated in typical picosecond regime computer simulations of protein dynamics in the solid state. The experimental root-mean-square angular fluctuations for both types of NH vectors increase to 37 degrees for viable wet cells at 10 degrees C. The similarity in mobilities for both general protein and cell wall peptidoglycan suggests that one additional motion in wet cells involves cooperative fluctuations of segments of cell walls, attached proteins, and associated cytoplasmic proteins.

Aromatic cross-links in insect cuticle: detection by solid-state 13C and 15N NMR.

Cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy has been used to determine insect cuticle composition and cross-link structure during sclerotization or tanning. Unsclerotized cuticle from newly ecdysed pupae of the tobacco hornworm, Manduca sexta L., had a high protein content with lesser amounts of lipid and chitin. Concentrations of chitin, protein, and catechol increased substantially as dehydration and sclerotization progressed. Analysis of intact cuticle specifically labeled with carbon-13 and nitrogen-15 revealed direct covalent linkages between ring nitrogens of protein histidyl residues and ring carbons derived from the catecholamine dopamine. This carbon-nitrogen adduct was present in chitin isolated from cuticle by alkaline extraction and is probably bound covalently to chitin. These data support the hypothesis that the stiffening of insect cuticle during sclerotization results primarily from the deposition of protein and chitin polymers and their crosslinking by quinonoid derivatives of catecholamines.

Solid-state NMR studies of Klebsiella pneumoniae grown under nitrogen-fixing conditions.

The carbon and nitrogen metabolism of Klebsiella pneumoniae M5a1 has been characterized using 13C and 15N labeling with detection by cross-polarization magic-angle spinning solid-state NMR. Cells grown on ammonium typically require some 20 h to derepress fully for nitrogenase when transferred to medium devoid of any source of fixed nitrogen. We have established that during this period some cellular proteins are catabolized with the liberated nitrogen being used for the synthesis of purines needed for formation of ribosomal RNA. The 20-h derepression period can be shortened to 6 h by the introduction of fixed nitrogen in certain specific forms. Serine is the most successful agent we have examined for shortening the derepression period and glycine among the least successful. We attribute this difference to the advantage of serine over glycine in providing both specific and nonspecific carbon and nitrogen sources for complete purine synthesis. These determinations were made by tracing the metabolism of 13C- and 15N-labeled chemical bonds from the 2 amino acids during derepression.

Solid-state NMR determination of glyphosate metabolism in a Pseudomonas sp.

The metabolism of the broad-spectrum herbicide, glyphosate (N-phosphonomethylglycine) in a soil Pseudomonas sp. PG2982 has been determined by cross-polarization magic-angle spinning 15N and 13C NMR of intact lyophilized cells. Using samples grown on 13C- and 15N-labeled glyphosate, we find that PG2982 does not metabolize glyphosate to aminomethylphosphonate as has been reported for mixed cultures of soil microbes. Rather, the phosphonomethyl carbon-nitrogen bond in glyphosate is cleaved, releasing glycine. Solid-state NMR analysis reveals that 20% of this glycine is used in the synthesis of purines, 35% is incorporated into protein as glycyl residues, with an additional 35% incorporated as seryl residues. The phosphonomethyl carbon of glyphosate is ultimately incorporated into a number of sites, including the C-2 and C-8 positions of the purine rings of nucleic acids, methyl groups of methionine and thymidine, and the methylene group of serine. The pattern of phosphonomethyl carbon incorporation indicates the involvement of tetrahydrofolate, a coenzyme which facilitates single-carbon transfers. This is the first complete determination of the metabolism of glyphosate in a pure culture, and the first bacterial metabolic study using both single and double cross-polarization solid-state NMR.

Lipid bilayer dynamics and rhodopsin-lipid interactions: new approach using high-resolution solid-state 13C NMR.

High-resolution, solid-state 13C NMR spectra have been obtained for unsonicated multilamellar dispersions of 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), recombinant membranes containing DLPC and rhodopsin, and native retinal rod disk membranes. The roles of 1H dipolar decoupling, 1H-13C cross-polarization, and magic-angle sample spinning have been investigated. Rotating-frame 13C relaxation times have been measured and are discussed in terms of lipid bilayer dynamics and rhodopsin-lipid interactions.

[N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture.

Solid-state [(15)N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium-assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen.

N- and [C]NMR determination of utilization of glycine for synthesis of storage protein in the presence of glutamine in developing cotyledons of soybean.

Solid-state (15)N- and [(13)C] NMR have been used to measure quantitatively the utilization of glycine in the presence of glutamine for the synthesis of storage protein in immature cotyledons of soybean (Glycine max L. cv. Elf) in culture. The presence of an equal molar amount of glycine in the medium causes a decrease in the use of glutamine-amide nitrogen. Glycine nitrogen is incorporated extensively into peptide bonds (in amounts greater than what would be expected if it appeared solely in glycine residues), but is used sparingly for synthesis of histidine ring residues, guanidino nitrogen residues of arginine, and lysine residues. The modest use of glycine carbon in protein synthesis does not parallel the use of glycine nitrogen.

Estimation of protein turnover in soybean leaves using magic angle double cross-polarization nitrogen 15 nuclear magnetic resonance.

Magic angle cross-polarization 15N nuclear magnetic resonance spectra of intact lyophilized soybean leaves have been obtained at 9.12 MHz. The leaves were harvested over a 6-week period from 15N-uniformly labeled plants exposed to 13CO2 for 1 to 7 days. The concentration of 13C-15N double labels in the main chains of leaf protein was determined nondestructively by double cross-polarization techniques. Both incorporation and turnover rates of these double labels were estimated and together lead to the conclusion that the protein of a mature, fully expanded soybean leaf is fairly long lived, with a half-life of about 30 days.

Asparagine amide metabolism in developing cotyledons of soybean.

Magic-angle single and double cross-polarization (13)C and (15)N NMR spectra have been obtained of lyophilized soybean cotyledons cultured on media containing, as the only nitrogen source, [4-(13)C, amide-(15)N]asparagine. Single cross-polarization NMR shows directly and unambiguously that both labels from asparagine are incorporated extensively and nonrandomly into protein by the developing cotyledon during a 2-week period. A stable-isotope double label tags a chemical bond. The metabolic fate of the asparagine double label was followed by double cross-polarization NMR using the latter's sensitivity to the dipolar coupling between directly bonded (13)C and (15)N. These experiments show that in culture the direct incorporation of asparagine (with no scrambling of label) accounts for about half of all asparagine residues in soybean protein. This conclusion implies the operation of a regulatory apparatus in soybeans for both direct utilization and degradation of asparagine in protein synthesis.

Characterization of photorespiration in intact leaves using carbon dioxide labeling.

The (13)C nuclear magnetic resonance spectra of sucrose extracted from intact soybean and corn leaves labeled with (13)CO(2) for 5 to 180 minutes have been obtained at 22.6 megahertz. The spectra provide a direct determination of the relative concentrations of (12)C-(13)C and (13)C-(13)C pairs of carbons in the labeled triose components of sucrose. This distribution of label is strongly dependent on the O(2) concentrations used during labeling for soybean but not for corn. Based on the time dependence of the experimental labeling patterns, photorespiration in air appears to be a sizable fraction of the apparent photosynthetic rate for soybean, but not for corn.

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

Fourier transform (13)C 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 (13)CO(2) 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.