13C nuclear magnetic resonance study of 17 alpha-substituted estradiols

We report the 13C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-gamma-lactone, phenyl, benzyl, naphthyl, etc.) at the 17 alpha-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12-18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17 alpha-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17 alpha-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments.     

Characterization of polyimides by 13C and 1H solid state nuclear magnetic resonance

The polyimides LaRC-TPI, LaRC-IA and LaRC-SI and related model compounds were investigated using 13C and 1H nuclear magnetic resonance (NMR). Magic angle spinning was used to acquire 13C isotropic chemical shift spectra of these materials. The spectra were assigned as completely as possible. In addition, the principal components of some shielding tensors were measured using variable angle correlation spectroscopy (VACSY). Of those studied, LaRC-SI is the only polymer that is soluble. However, after it is heated past its glass transition temperature, LaRC-SI becomes insoluble. Experiments were performed in an attempt to identify causes of this behavior. When magnetization from nuclei in rigid regions of the polymer is suppressed, the 1H and 13C NMR spectra of soluble LaRC-SI are significantly different from those of insoluble LaRC-SI. Hydration studies of LaRC-SI show that absorbed water plasticizes the polymer.     

Solid-state 13C nuclear magnetic resonance spectra of 6-aminopenicillanic acid

Factors which affected the severity of scoliosis induced by rib resection were examined. Seventy chickens were divided into a rib transection group and a rib resection group with seven subgroups, according to the number of ribs treated and the age at surgery, and examined radiographically until 20 weeks postoperatively. Rib transection group chickens showed fusion at the transected site and no marked scoliosis. In the rib resection group, the more ribs resected and the younger the age at operation, the more severely the scoliosis developed. Bone regeneration at the resection site made the progression of scoliosis milder. These findings indicate that the age at rib resection, the number of ribs that are operated on, and nonunion of the resected sites play important roles in the progression of scoliosis induced by rib resection.     

13C nuclear magnetic resonance study of molecular motions and conformational transitions in muscle calcium binding parvalbumins

13C nuclear magnetic resonance is used to detect the Ca2+ ion controlled conformational transition in muscle calcium binding parvalbumin and to study its intramolecular motions. Nuclear relaxation parameters are used to evaluate the reorientation rates of the protein and some of the amino acid side chains. While peripheral residues exhibit greater motional freedom than the protein interior, an interesting finding is that significant rapid internal motion is present in the phenylalanine rings comprising the hydrophobic core of the protein.     

1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.     

Biosynthesis of bikaverin in Fusarium oxysporum. Use of 13C nuclear magnetic resonance with homonuclear 13C decoupling to locate adjacent 13C labels

Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.     

Use of 13C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, 13C nuclear magnetic resonance (NMR) performed with uniformly enriched [13C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the 13C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

A study of precipitation using nuclear magnetic resonance: Cu-13 at.% Be

We examine the nuclear magnetic resonance (nmr) intensity of one of the components in one of the phases in a two phase binary alloy undergoing precipitation. It is shown that for certain nuclei one can readily follow the course of the reaction, and ideally can determine quantitatively the composition and amount of the final matrix if the precipitate composition is known. We develop expressions relating the resonance intensity to the degree of completion of the reaction for

• 1.(a) discontinuous and
• 2.(b) continuous precipitation.

These are used to interpret the intensity vs ageing time at 280°C for a Cu-13 at.% Be alloy. The ratio of the intensity of the ⁶³Cu in the matrix in the fully aged alloy to that in the as quenched alloy is also examined and discussed in light of our experimental results. It is argued that interfacial effects at phase boundaries cause significant deviations from expected intensities.     

Use of in vivo 13C nuclear magnetic resonance spectroscopy to follow sugar uptake in Zymomonas mobilis

An image analyzer was applied to pre- and post-embedding immunogold labeling with 5-nm gold probes in electron micrographs of several skin basement membrane antigens to improve the visualization of immunolabeling. With a TV camera connected to a color image analyzer, an image of an original immunoelectron micrograph was projected on a TV screen. The image was recorded in the analyzer as Record 1. After the floating threshold method procedure to reduce the contrast of the skin structure, electron-dense 5-nm gold particles could be easily detected. With the analyzer, these particles were then suitably enhanced in color and in size and their image was recorded as Record 2. Records 1 and 2 were then overlapped on the TV screen to build up a double-image picture. Compared with the small, electron-dense 5-nm gold particles in the original electron micrograph, ultrastructural localization of bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and the collagenous part of Type VII collagen were more clearly and easily seen, even on low-magnification electron micrographs. The level of background labeling could also be accurately and objectively evaluated. By counting the number of gold particles labeling a certain area and using the analyzer to interpret the result as a diagram, quantitative analysis was also possible. We believe that this technique should be widely applicable to immunogold electron microscopy, not only of skin but also of other substrates of interest.     

Studies on the stereoselective internal acyl migration of ketoprofen glucuronides using 13C labeling and nuclear magnetic resonance spectroscopy

BACKGROUND: To improve asthma disease management, the National Asthma Education Program (NAEP) Expert Panel published Guidelines for the Diagnosis and Management of Asthma in 1991. OBJECTIVES: To compare the current status of asthma disease management among patients in a large health maintenance organization with the NAEP guidelines and to identify the factors that may be associated with medical care (eg, emergency department visits and hospital admissions) and adherence to the guidelines. METHODS: Analyses of 1996 survey data from 5580 members with asthma (age range, 14 to 65 years) covered by a major health maintenance organization in California (Health Net). RESULTS: In general, adherence to NAEP guidelines was poor. Seventy-two percent of respondents with severe asthma reported having a steroid inhaler, and of those, only 54% used it daily. Only 26% of respondents reported having a peak flowmeter, and of those, only 16% used it daily. Age (older), duration of asthma (longer), increasing current severity of disease, and treatment by an asthma specialist correlated with daily use of inhaled steroids. Ethnicity (African American and Hispanic) correlated negatively with inhaled steroid use but positively with emergency department visits and hospital admissions for asthma. Increasing age and treatment by an asthma specialist were also identified as common factors significantly related to the daily use of a peak flowmeter and, interestingly, to overuse of beta2-agonist metered-dose inhalers. CONCLUSIONS: Although the NAEP guidelines were published 7 years ago, compliance with the guidelines was low. It was especially poor for use of preventive medication and routine peak-flow measurement. Furthermore, the results showed that asthma specialists provided more thorough care than did primary care physicians in treating patients with asthma. Combining the results of the regression analyses revealed that some of the variation in rates of emergency department visits and hospitalizations among some subpopulations can be explained by the underuse of preventive medication. This study serves the goal of documenting the quality of care and services currently provided to patients with asthma through a large health maintenance organization and provides baseline information that can be used to design and assess effective population-based asthma disease management intervention programs.     

1H nuclear magnetic resonance study on equilibrium between two four-stranded solution conformations of short d(CnT)

NMR analysis of d(C4T) showed the slow exchange between two distinct tetramers (each fully symmetric) in solution. For one tetramer, NOE cross-peak patterns characteristic of an i-motif structure (H1'-H1' and H6-H1'/H1'-H6) were observed between C1 and T5, indicating that this tetramer takes a completely intercalated conformation where the T5 residue is stacked on the C1.C1(+) pair of the other duplex (S-form). The other was found to be a tetramer in which one of the duplexes is shifted by one nucleotide unit (R-form), resulting in nonstacking 3' end thymidine residues and an equal number of stacked C.C+ pairs to that of the S-form. The same spectral features were observed for d(C3T) but neither for d(TC3) nor d(TC4), indicative of the critical role of the position of the thymidine residue in the tetrad isomerization. From NMR denaturation profiles, the S-forms were found to be more stable than the R-forms, and the linear relationship between the logarithm of the equilibrium constant (K = [tetramer]/[single]4) and the inverse of temperature (1/T) was confirmed for both forms, indicating conformity to the two-state transition model. Both enthalpy and entropy values of the formation of the S-form from four single strands were more negative than those of the R-form. The enthalpy term should contribute to the stabilization of the S-forms at low temperatures. The difference of the free energy values [DeltaG degrees(S-form) - DeltaG degrees(R-form)] was found to be -2.1 and -2.7 kJ.mol-1 at 20 degreesC for d(C4T) and d(C3T), respectively, explaining the higher stability of the S-forms. With increasing temperature, these two topologies were found to comparably exist at equilibrium in solution with slow exchange via dissociation to the single strands. A biological role of this topological isomerization is also suggested.     

Kinetics of binding of methyl alpha- and beta-D-galactopyranoside to peanut agglutinin: a carbon-13 nuclear magnetic resonance study

The binding kinetics of methyl alpha- and methyl beta-D-galactopyranoside to the anti-T lectin from peanuts were studied by 13C NMR, employing methyl galactopyranosides specifically enriched in 13C at C-1. Association and dissociation rate constants, as well as their activation parameters, are reported. The association rate constants, 4.6 X 10(4) M-1 s-1 for the alpha-galactopyranoside and 3.6 X 10(4) M-1 s-1 for the beta-galactopyranoside, are several orders of magnitude below those expected for a diffusion-controlled process. For both anomers, the association rate constant was temperature independent, implying that the association process occurs without a significant activation enthalpy. However, a considerable association activation entropy was found for both ligands. The dissociation rate constants were in the range of 9-46 s-1 within a temperature range of 5-35 degrees C for the alpha-galactopyranoside, and in the range of 9-39 s-1 within a temperature range of 5-25 degrees C for the beta-galactopyranoside. A considerable dissociation activation enthalpy of ca. 10 kcal mol-1 was found for both anomers. A two-step binding model, consistent with the present NMR data and with previous UV and CD spectroscopic data, is presented.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-cyano, 17 alpha-aminomethyl, and 17 alpha-alkylamidomethyl derivatives of 5 alpha-dihydrotestosterone and testosterone

17 alpha-Aminomethyl, 17 alpha-acetamidomethyl, and 17 alpha-hemiglutaramidomethyl derivatives of dihydrotestosterone and testosterone have been prepared by hydrocyanation of 3,3'-(ethylenedioxy)-5 alpha-androstan-17-one and 3,3'-ethylenedioxyandrost-5-en-17-one, reduction of the corresponding acetylated 17 alpha-cyanohydrins with lithium aluminium hydride, and acylation of the resulting 17 alpha-aminomethyl derivatives with either acetic anhydride or the mono acid chloride of glutaric acid mono methyl ester. Saponification of the 17 alpha-hemiglutaramidomethyl methyl esters gave the corresponding hemiglutaramido derivatives, while acid hydrolysis of the 3-ethylene ketal group of 17 alpha-acetamidomethyl and 17 alpha-hemiglutaramidomethyl derivatives regenerated the 3-oxo and 3-oxo-4-ene functions. The 17 alpha-configuration of 17-substituted steroids was determined by 1H and 13C NMR and confirmed by comparing with NMR data for 17 alpha- and 17 beta-cyano-17-hydroxyandrost-4-en-3-one, 17 beta-cyano-3,3'-(ethylenedioxy)androst-5-en-17-ol, 17 alpha-alkynyl, and 17 alpha-hexanoic derivatives of dihydrotestosterone and testosterone, of known 17-configurations. Several ambiguous assignments of 13C NMR signals of 17 alpha-substituted steroids and unsubstituted 17 beta-hydroxy or 17-oxo precursors have been resolved using steroid analogs deuterated at positions C5-7, or C16 for androstane derivatives, and at positions C6-7, or C7 for androstene derivatives. 17 alpha-Aminomethyl and 17 alpha-alkylamidomethyl derivatives of dihydrotestosterone and testosterone are useful intermediates for the access to potential ligands of androgen-binding proteins necessary for affinity chromatography purification or affinity-labeling experiments.     

Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of D-[1-13C]glucose metabolism

Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, gamma-aminobutyrate (GABA), glutamine, and aspartate. D-[1-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C-NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbonatom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates.(ABSTRACT TRUNCATED AT 250 WORDS)