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The effect of the enrichment of sunflower oil with gamma-tocopherol on the evolution of its oxidation is the subject of this study. Sunflower oil and samples of this oil enriched with different gamma-tocopherol concentrations are submitted to accelerated storage conditions and their evolution, until total polymerization is monitored by H-1 NMR spectroscopy. The concentrations of linoleic acyl groups, of primary and of a great number of secondary oxidation compounds are determined throughout the process. These concentrations, and the times at which the main changes in them occur are analyzed to elucidate if they are valid markers of the global effect of gamma-tocopherol enrichment. It is proved for the first time that certain comparisons between concentrations of some of the compounds formed in oxidation can lead to erroneous conclusions about the effect of this enrichment. It is shown that, during a first short period of time under accelerated storage conditions, the higher the concentration of gamma-tocopherol the faster the degradation of linoleic acyl groups and the higher the concentration of hydroperoxides; after this first period this relationship is more complex. Generally, the higher the gamma-tocopherol concentration the slower the degradation of the oil, except in the first period.Practical Applications: This study provides very valuable information about the effect caused by enrichment with gamma-tocopherol on sunflower oil oxidative stability and oxidation across a very broad range of concentrations. In addition, the amount of information obtained can be of great importance from several points of view because the oxidation process has been monitored globally. This may be of interest not only to researchers involved in the analysis of oxidation mechanisms but also to those in the field of technology and edible oil legislation, and even to those worried about edible oil safety and its effect on nutrition and health. Another important feature of this study, from the practical point of view, is the methodology used because it does not involve the chemical modification of the sample and the same H-1 NMR spectrum is able to provide information about all the above-mentioned aspects. Sunflower oil enriched with different levels of gamma-tocopherol. Concentrations of primary and secondary oxidation compounds after 4 and 9 days, respectively, under accelerated storage conditions. This work has been supported by the Spanish Ministry of Economy and Competitiveness (MINECO AGL 2015-65450-R), the Basque Government (EJ-GV, IT-916-16), and the Agriculture Department of the Basque Government (PA19/02).

Two‐component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K+ homeostasis. In Escherichia coli, the histidine kinase KdpD senses K+ availability, whereas the response regulator KdpE activates synthesis of the high‐affinity K+ uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate‐responsive two‐component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two‐hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross‐regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K+ and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K+ and phosphate homeostasis and provides a mechanism for fine‐tuning of the balance between positively and negatively charged ions in the bacterial cell.

Summary Biomacromolecules in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of biomolecules, in particular proteins, are extended for several angstroms. The role of the hydration shells around a protein has yet to be completely understood. Hydrated proteins maintain more dynamic flexibility with respect to the dried system, which is an important property in protein-protein and/or protein-ligand recognition processes. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. The water proton spin-lattice relaxation rate in protein solution is analyzed considering all possible dipolar contributions from coupled protons environments. The analysis of both selective and non-selective water spin-lattice relaxation rates allowed the calculation of the average effective correlation time for the water molecules at the protein interface and the evaluation of the long range ordering effect of the protein surface.

The previous work on the hydrolysis and alcoholysis reactions of phosphonium ylides is summarized and reviewed in the context of their currently accepted mechanisms. Several experimental facts relating to ylide hydrolysis and to salt and ylide alcoholysis are shown to conflict with those mechanisms. In particular, we demonstrate that the pK(a) values of water and alcohols are too high in organic media to bring about protonation of ylide. Therefore, we propose concerted addition of the water or alcohol O-H bond across the ylide P=C bond. In support of this, we provide NMR spectroscopic evidence for equilibrium between ylide and aclohol that does not require the involvement of phosphonium hydroxide. We report the first P-alkoxyphosphorane to be characterised by NMR spectroscopy that does not undergo exchange on an NMR timescale. Two-dimensional NMR spectroscopic techniques have been applied to the characterisation to P-alkoxyphosphoranes for the first time.

A series of sodium borosilicate glasses were synthesized with increasing fractions of KReO4 or Re2O7, to 10000 ppm (1 mass%) target Re in glass, to assess the effects of large concentrations of rhenium on glass structure and to estimate the solubility of technetium, a radioactive component in typical low active waste nuclear waste glasses. Magic angle spinning nuclear magnetic resonance (MAS-NMR), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy were performed to characterize the glasses as a function of Re source additions. In general, silicon was found coordinated in a mixture of Q2 and Q3 structural units, while Al was 4-coordinated and B was largely 3-coordinate and partially 4-coordinated. The rhenium source did not appear to have significant effects on the glass structure. Thus, at the up to the concentrations that remain in dissolved in glass, ~3000 ppm Re by mass maximum. , the Re appeared to be neither a glass-former nor a strong glass modifier., Rhenium likely exists in isolated ReO4- anions in the interstices of the glass network, as evidenced by the polarized Raman spectrum of the Re glass in the absence of sulfate. Analogous to SO42-¬ in similar glasses, ReO4- is likely a network modifier and formsmore » alkali salt phases on the surface and in the bulk glass above solubility.« less

"Several factors affect antenna-soil coupling in a Ground Penetrating Radar (GPR) survey, like surface. roughness, lithology, lateral heterogeneities, vegetation, antenna height from the surface and. water content. Among them, lithology and water content have a direct effect on the bulk electromagnetic. properties of the material under investigation. It has been recently pointed out that the wavelet. of the early-time portion of a radar signal is correlated to the shallow subsurface dielectric properties. of a material. This result indicates that some information on such properties can be directly. extracted from the analysis of GPR early-time traces.. In the present paper, we use the early-time GPR signal, in terms of average envelope amplitude. computed on the first half-cycle, to map the near-surface (few centimetres) lateral distribution of. dielectric parameters, induced by changing the shallow water content on a concrete slab. This controlled. experiment was specifically designed to study the effect of water content variations on. antenna-material coupling, minimizing the influence of both surface roughness and heterogeneity.. The quantitative control of the water in the shallow portion of the slab is performed by using a portable. unilateral Nuclear Magnetic Resonance (NMR) sensor, which is able to determine the water. content in the material on the basis of the measured proton density. The results show a matching. pattern of the physical parameters measured with the two different techniques and a very high. degree of linear correlation (r = 0.97) between the radar early-time signal average amplitude and the. intensity of the NMR signal, which is proportional to the proton density, i.e., to the water content.. This experiment suggests that the early-time approach could be used as a fast and high- spatial. resolution tool for qualitatively mapping water content lateral variations in a porous material at. shallow depth, using a ground-coupled single-offset antenna configuration and that a quantitative. evaluation of the moisture content would require a calibration procedure."

The ability for a biofilm to grow and function is critically dependent on the nutrient availability, and this in turn is dependent on the structure of the biofilm. This relationship is therefore an important factor influencing biofilm maturation. Nutrient transport in bacterial biofilms is complex; however, mathematical models that describe the transport of particles within biofilms have made three simplifying assumptions: the effective diffusion coefficient (EDC) is constant, the EDC is that of water, and/or the EDC is isotropic. Using a Monte Carlo simulation, we determined the EDC, both parallel to and perpendicular to the substratum, within 131 real, single species, three-dimensional biofilms that were constructed from confocal laser scanning microscopy images. Our study showed that diffusion within bacterial biofilms was anisotropic and depth dependent. The heterogeneous distribution of bacteria varied between and within species, reducing the rate of diffusion of particles via steric hindrance. In biofilms with low porosity, the EDCs for nutrient transport perpendicular to the substratum were significantly lower than the EDCs for nutrient transport parallel to the substratum. Here, we propose a reactiondiffusion model to describe the nutrient concentration within a bacterial biofilm that accounts for the depth dependence of the EDC. Biotechnol. Bioeng. 2012; 109:12801292. (C) 2011 Wiley Periodicals, Inc.

Structural knowledge of interactions amongst the similar to 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Forster resonance energy transfer (FRET) was used to investigate the 10 mu M K-D (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.

Proton nuclear magnetic resonance (¹H-NMR) relaxometry was used to study the effects of high pressure and thermal processing on membrane permeability and cell compartmentalization, important components of plant tissue texture. High pressure treated onions were subjected to pressure levels from 20 to 200 MPa at 5 min hold time at initial temperatures of 5 and 20 °C. Thermally treated onions were exposed for 30 min at temperatures from 40 to 90 °C. Loss of membrane integrity was clearly shown by changes in transverse relaxation time (T(2)) of water at temperatures of 60 °C and above. Destabilization effects on membranes exposed to high pressure were observed at 200 MPa as indicated by T(2) measurements and cryo-scanning electron microscopy (Cryo-SEM). T(2) relaxation successfully discriminated different degrees of membrane damage based on the T(2) shift of the vacuolar component. Analyses of the average water self-diffusion coefficient indicated less restricted diffusion after membrane rupture occurred in cases of severe thermal treatments. Milder processing treatments yielded lower average diffusion coefficients than the controls. ¹H-NMR proved to be an effective method for quantification of cell membrane damage in onions and allowed for the comparison of different food processes based on their impact on tissue integrity.

According to previous reports, Lactococcus lactis imports glucose via two distinct phosphoenolpyruvate: phosphotransferase systems (mannose-PTS and cellobiose-PTS) and one or more unknown non-PTS permease(s). GlcU was identified as the sole non-PTS permease involved in the transport of glucose. Additionally, the biochemical properties of PTS(Man), PTS(Cel) and GlcU were characterized in double knockout mutants with glucose uptake restricted to a single system. Transport susceptibility to protonophores indicated that glucose uptake via GlcU is proton-motive force dependent. Competition assays revealed a high specificity of GlcU for glucose. Furthermore, the permease has low affinity for glucose and displays strong preference for the beta-anomer as shown by the profiles of consumption of the two glucose anomers studied by (13)C-NMR. Similar kinetic properties were found for PTS(Cel), while PTS(Man) is a high-affinity system recognizing equally well the two anomeric forms of glucose. Transcripts of the genes encoding the three transporters are present simultaneously in the parent strain NZ9000 as shown by reverse transcription-PCR. Investigation of the distribution of GlcU homologues among bacteria showed that these proteins are restricted to the low-GC Gram-positive Firmicutes. This work completes the identification of the glucose transport systems in L. lactis MG1363.

Conformational analysis of angiotensin I (AI) and II (AII) peptides has been performed through 2D (1) H-NMR spectroscopy in dimethylsulfoxide and 2,2,2-trifluoroethanol/H-2 O. The solution structural models of AI and AII have been determined in dimethylsulfoxide using NOE distance and (3) J (HNHalpha) coupling constants. Finally, the AI family of models resulting from restrained energy minimization (REM) refinement, exhibits pairwise rmsd values for the family ensemble 0.26 +/- 0.13 Angstrom, 1.05 +/- 0.23 Angstrom, for backbone and heavy atoms, respectively, and the distance penalty function is calculated at 0.075 +/- 0.006 Angstrom(2) . Comparable results have been afforded for AII ensemble (rmsd values 0.30 +/- 0.22 Angstrom, 1.38 +/- 0.48 Angstrom for backbone and heavy atoms, respectively; distance penalty function is 0.029 +/- 0.003 Angstrom(2) ). The two peptides demonstrate similar N-terminal and different C-terminal conformation as a consequence of the presence/absence of the His9-Leu10 dipeptide, which plays an important role in the different biological function of the two peptides. Other conformational variations focused on the side-chain orientation of aromatic residues, which constitute a biologically relevant hydrophobic core and whose inter-residue contacts are strong in dimethylsulfoxide and are retained even in mixed organic-aqueous media. Detailed analysis of the peptide structural features attempts to elucidate the conformational role of the C-terminal dipeptide to the different binding affinity of AI and AII towards the AT(1) receptor and sets the basis for understanding the factors that might govern free- or bound-depended AII structural differentiation. European Journal of Biochemistry

Summary • Changes in the physical state of cellular water and its interrelations with carbohydrate metabolism were studied during preplanting storage of tulip bulbs (Tulipa gesneriana‘Apeldoorn’). • Magnetic resonance imaging, light and scanning electron microscopy and high-performance anion exchange chromatography with pulsed amperometric detection were used to follow time-dependent changes during bulb storage at 17 or 20°C (nonchilled) or 4°C (chilled). • No visible differences in scale structure and central bud development were observed microscopically between chilled and nonchilled bulbs. However, the scales of the chilled bulbs exhibited higher water content, faster starch degradation and increased concentrations of sucrose and ethanol-soluble fructan. Quantitative measurements of magnetization transfer (MT) indicated a smaller fraction of a solid or a restricted-mobility proton pool in the scales of the chilled bulbs. By contrast, the MT effect was significantly higher in the central bud of the chilled than in the nonchilled bulbs. • Degradation of storage polysaccharides to low-molecular-weight sugar molecules during release from dormancy could be accompanied by local release of water molecules tightly bound to the polysaccharide granules into the bulk water, or by an influx of free water molecules due to increased osmotic potential caused by the raised sugar concentration, or by a combination of both effects.

The coupling of high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high held magnets have given a new stimulus to this technique, which has emerged since the mid 1990s as a very efficient method for the on-line identification of organic molecules. LC-NMR thus represents a potentially interesting complementary technique to LC-UV-MS in phytochemical analysis for the detailed on-line structural analysis of natural products. Recent applications have fully demonstrated the usefulness of this technique. A brief review of the applications of LC-NMR in natural product chemistry is presented in this paper, and a summary of the basic principles and modes of operation of LC-NMR is provided. Selected examples of LC-NMR analyses of plant metabolites in crude extracts or in enriched fractions are outlined and used to illustrate the different strategies for employing the technique. The practical possibilities and limitations of LC-MMR in its application to the analysis of crude plant extracts are discussed by means of several examples. Analytical strategies involving LC multi-coupled (hyphenated) techniques for the chemical screening and dereplication of crude plant extracts are presented. An analysis of the future development of the technique with respect to its application in photochemical analysis is also given. Copyright (C) 2001 John Wiley & Sons, Ltd.

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N',N"-triacetylchitotriose has been analyzed by H-1-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target tee solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N,N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 Angstrom, whereas the heavy atom RMSD was 2.18 Angstrom. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyrsubstitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains, The experimentally derived structures and the? values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems. (C) 2000 Wiley-Liss, Inc.

A method for in vivo carbon-edited detection with proton echo-planar spectroscopic imaging (ICED PEPSI) is described. This method is composed of an echo-planar based acquisition implemented with C-13-H-1 J editing spectroscopy and is intended for high temporal and spatial resolution in vivo spectroscopic imaging of C-13 turnover, from D-[1,6-C-13]glucose to glutamate and glutamine, in the brain. At a static magnetic field strength of 7 T, both in vitro and in vivo chemical shift imaging data are presented with a spatial resolution of 8 mu L (i.e., 1.25 x 1.25 x 5.00 mm(3)) and a maximum spectral bandwidth of 5.2 ppm in H-1. Chemical shift imaging data acquired every 11 minutes allowed detection of regional [4-(CH2)-C-13]glutamate turnover in rat brain. The [4-(CH2)-C-13]glutamate turnover curves, which can be converted to tricarboxylic acid cycle fluxes, showed that the tricarboxylic acid cycle flux (V-TCA) in pure gray and white matter can range from 1.2 +/- 0.2 to 0.5 +/- 0.1 mu mol/g/min, respectively, for morphine-anesthetized rats. The mean cortical V-TCA from 32 voxels of 1.0 +/- 0.3 mu mol/g/min (N = 3) is in excellent agreement with previous localized measurements that have demonstrated that V-TCA can range from 0.9-1.1 mu mol/g/min under identical anesthetized conditions. (C) 1999 Wiley-Liss, Inc.

Protein phosphorylation is one of the major signal transduction mechanisms for controlling and regulating intracellular processes, Phosphorylation of specific hydroxylated amino acid side chains (Ser, Thr, Tyr) by protein kinases can activate numerous enzymes; this effect can be reversed by the action of protein phosphatases. Here we report ab initio (HF/6-31G* and Becke3LYP/6-31G*) and semiempirical (PM3) molecular orbital calculations pertinent to the ion pair formation of the phosphorylated amino acids with the basic side chains of Lys and Arg, Methyl-, ethyl-, and phenylphosphate, as well as methylamine and methylguanidinium were used as model compounds for the phosphorylated and basic amino acids, respectively. Phosphorylated amino acids were calculated as mono- and divalent anions, Our results indicate that the PSer/PThr ion pair interaction energies are stronger than those with PTyr, Moreover, the interaction energies with the amino group of Lys are generally more favorable than with the guanidinium group of Arg, The Lys amino groups form stable bifurcated hydrogen bonded structures; while the Arg guanidinium group can form a bidentate hydrogen bonded structure, Reasonable values for the interaction free energies in aqueous solution were obtained for some complexes by the inclusion of a solvent reaction field in the computation (PM3-SM3). (C) 1996 Wiley-Liss, Inc.

Bombolitin II is a member of a family of five structurally related heptadecapeptides, originally isolated from the venom of a bumblebee, acting at the membrane level and able to increase the activity of phospholipase A 2 . The biological activity of bombolitins seems to be related to their ability to form amphipathic helical structures. To complete our systematic studies on this class of peptides, in the present work we synthesized bombolitin II by solid phase methodology. The secondary structure of this peptide was fully characterized by circular dichroism, 1 H NMR, distance geometry and molecular dynamics simulation. The peptide tends to aggregate in aqueous solution at millimolar concentrations, with formation of bundles of amphiphilic helices. In dilute aqueous solution (10 -5 mol/L) bombolitin II adopts an amphipathic helical structure in the presence of sodium dodecyl sulfate. The helical segment spans the sequence from residue 2 to residue 16. The results are in line with the hypothesis that this class of peptides modulate the acitivity of phospholipase A 2 by altering the physical state of the aggregated substrate.

PURPOSE: Measurement of the cerebral metabolic rate of oxygen (CMRO2 ) by means of direct imaging of the 17 O signal can be a valuable tool in neuroscientific research. However, knowledge of the longitudinal and transverse relaxation times of different brain tissue types is required, which is difficult to obtain because of the low sensitivity of natural abundance H2 17 O measurements. METHODS: Using the improved sensitivity at a field strength of 16.4 Tesla, relaxation time measurements in the rat brain were performed in vivo and postmortem with relatively high spatial resolutions, using a chemical shift imaging sequence. RESULTS: In vivo relaxation times of rat brain were found to be T1 = 6.84 +/- 0.67 ms and T2 * = 1.77 +/- 0.04 ms. Postmortem H2 17 O relaxometry at enriched concentrations after inhalation of 17 O2 showed similar T2 * values for gray matter (1.87 +/- 0.04 ms) and white matter, significantly longer than muscle (1.27 +/- 0.05 ms) and shorter than cerebrospinal fluid (2.30 +/- 0.16 ms). CONCLUSION: Relaxation times of brain H2 17 O were measured for the first time in vivo in different types of tissues with high spatial resolution. Because the relaxation times of H2 17 O are expected to be independent of field strength, our results should help in optimizing the acquisition parameters for experiments also at other MRI field strengths. Magn Reson Med, 2015. (c) 2015 Wiley Periodicals, Inc.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-II(mtl).The backbone resonances were partially assigned using three-dimensional heteronuclear H-1 NOE H-1-N-15 single-quantum coherence (N-15 NOESY-HSQC) spectroscopy and three-dimensional heteronuclear H-1 total correlation H-1-N-15 single-quantum coherence (N-15 TOCSY-HSQC) spectroscopy on uniformly ''N enriched protein. Triple-resonance experiments on uniformly N-15/C-13 enriched protein were necessary to complete the backbone assignments, due to overlapping H-1 and N-15 frequencies. Data obtained from three-dimensional H-1-N-15-C-13alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional H-1-N-15-(CO)-C-13 correlation experiment (HNCO), and a three-dimensional H-1alpha-C-13alpha-(CO)-C-13 correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances.Determination of the secondary structure of IIA(mtl) is based upon NOE information from the N-15 NOESY-HSQC and the H-1alpha and C-13alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIA(glc) of E. coli and Bacillus subtilis determined by NMR and X-ray.