Combining pure shift and J-edited spectroscopies: A strategy for extracting chemical shifts and scalar couplings from highly crowded proton spectra of oligomeric saccharides.

We report the application of pure shift and J-edited nuclear magnetic resonance spectroscopies to the structural analysis of a protected maltotrioside synthetic intermediate whose crowded 1 H spectrum displays highly crowded regions. The analytical strategy is based on the implementation of J-edited and TOCSY experiments whose resolution is optimized by the use of broadband homonuclear decoupling and selective refocusing techniques, to assign and measure chemical shifts and homonuclear scalar couplings with high accuracy. The resulting data show a high level of complementarity, providing a detailed insight into each subunit of this oligomeric saccharide, even for proton sites whose nuclear magnetic resonance signals strongly overlap. This approach allowed for fully assigning proton chemical shifts and extracting 80% of the 3 JHH couplings that are in excellent agreement with those expected for D-gluco-pyranosyl units in 4 C1 conformations.

Magnetic field dependence of spatial frequency encoding NMR as probed on an oligosaccharide.

The magnetic field dependence of spatial frequency encoding NMR techniques is addressed through a detailed analysis of (1)H NMR spectra acquired under spatial frequency encoding on an oligomeric saccharide sample. In particular, the influence of the strength of the static magnetic field on spectral and spatial resolutions that are key features of this method is investigated. For this purpose, we report the acquisition of correlation experiments implementing broadband homodecoupling or J-edited spin evolutions, and we discuss the resolution enhancements that are provided by these techniques at two different magnetic fields. We show that performing these experiments at higher field improves the performance of high resolution NMR techniques based on a spatial frequency encoding. The significant resolution enhancements observed on the correlation spectra acquired at very high field make them valuable analytical tools that are suitable for the assignment of (1)H chemical shifts and scalar couplings in molecules with highly crowded spectrum such as carbohydrates.

Limit of miscibility and nanophase separation in associated mixtures.

We present a detailed analysis of the mixing process in an associating system, the water-tert-butanol (2-methyl-2-propanol) mixture. Using molecular dynamics simulations, together with neutron, X-ray diffraction experiments, and pulsed gradient spin-echo NMR, we study the local structure and dynamic properties over the full concentration range, and thereby provide quantitative data that reveal relationships between local structure and macroscopic behavior. For an alcohol-rich mixture, diffraction patterns from both neutron and X-ray experiments exhibit a peak at low wavelength vector (q ≈ 0.7 Å(-1)) characteristic of supermolecular structures. On increasing the water content, this "prepeak" progressively flattens and shifts to low wave vector . We identify hydrogen bonds in the system as the driving force for the specific organization that appears in mixtures, and provide an analysis of the variation of the cluster size distribution with composition. We find that the sizes of local hydrogen-bonded clusters observed in alcohol-rich mixtures become larger as the mole fraction, x(w), of water is increased; a nanophase separation is seen for x(w) in the range 0.6-0.7. This corresponds to several changes in some macroscopic properties of the liquid mixture. Thus, we propose a microscopic description of the effect of water addition in alcohol, which is in agreement with both neutron diffraction pattern and mobility of water and alcohol species. In summary we present a full and comprehensive description of miscibility at its limit in an associated system.

Improvements to selective refocusing phased (SERFph) experiments.

Selective refocusing experiments are very powerful for extracting proton-proton couplings one by one. However we demonstrate in the present work that various spectral artefacts are produced by the initial sequence and we show that the combined addition of a refocusing pi pulse and a zero-quantum filter greatly improves the experimental sensitivity, and moreover leads to observation of pure absorption lineshapes in the resulting phased 2D spectrum. These developments are applied to the differentiation of enantiomers dissolved in a chiral liquid crystal.

Selenium bioavailability and uptake as affected by four different plants in a loamy clay soil with particular attention to mycorrhizae inoculated ryegrass.

The aim of this study was to investigate the influence of plant species, especially of their rhizosphere soil, and of inoculation with an arbuscular mycorrhizal (AM) fungus on the bioavailability of selenium and its transfer in soil-plant systems. A pot experiment was performed with a loamy clay soil and four plant species: maize, lettuce, radish and ryegrass, the last one being inoculated or not with an arbuscular mycorrhizal fungus (Glomus mosseae). Plant biomass and Se concentration in shoots and roots were estimated at harvest. Se bioavailability in rhizosphere and unplanted soil was evaluated using sequential extractions. Plant biomass and selenium uptake varied with plant species. The quantity of rhizosphere soil also differed between plants and was not proportional to plant biomass. The highest plant biomass, Se concentration in plants, and soil to plant transfer factor were obtained with radish. The lowest Se transfer factors were obtained with ryegrass. For the latter, mycorrhizal inoculation did not significantly affect plant growth, but reduced selenium transfer from soil to plant by 30%. In unplanted soil after 65 days aging, more than 90% of added Se was water-extractable. On the contrary, Se concentration in water extracts of rhizosphere soil represented less than 1% and 20% of added Se for ryegrass and maize, respectively. No correlation was found between the water-extractable fraction and Se concentration in plants. The speciation of selenium in the water extracts indicated that selenate was reduced, may be under organic forms, in the rhizosphere soil.

Visualisation of enantiomers via insertion of a BIRD module in X-H correlation experiments in chiral liquid crystal solvent.

Several 13C-1H NMR techniques are derived simplifying the visualisation of enantiomers in chiral ordering solvents. They proceed through various heteronuclear 2D experiments where a bilinear rotation decoupling sequence (BIRD) is inserted in the middle of the t1 evolution period. In this way, the small couplings are refocused while the large couplings are preserved. The methods allow extracting precise values of one-bond carbon-proton residual dipolar couplings for each enantiomer out of unresolved proton-coupled 13C or carbon-coupled 1H spectra. Illustrative examples are analysed and discussed using various pulse sequences.

An investigation of the structure and bond rotational potential of some fluorinated ethanes by NMR spectroscopy of solutions in nematic liquid crystalline solvents.

NMR spectra of 1,2-dibromo-1,1-difluoroethane and 1-bromo-2-iodo-tetrafluoroethane dissolved in nematic liquid crystalline solvents have been analysed to yield the magnitudes and signs of the scalar couplings, J(ij), and total anisotropic couplings, T(ij), between all the (1)H, (19)F, and (13)C nuclei, except for those between two (13)C nuclei. The values obtained for T(ij) in principle contain a contribution from J(ij)(aniso), the component along the static applied magnetic field of the anisotropic part of the electron-mediated spin-spin coupling. Neglecting this contribution allows partially averaged dipolar couplings, D(ij), to be extracted from the T(ij), and these were used to determine the structure, orientational order, and the conformational distribution generated by rotation about the C-C bond. The values obtained are compared with the results of calculations by ab initio and density functional methods. The differences found are no greater than those obtained for similar compounds which do not contain fluorine, so that there is no definitive evidence for significant contributions from J(CF)(aniso) or J(FF)(aniso) in the two compounds studied.

Obtaining the structure and bond rotational potential of a substituted ethane by NMR spectroscopy of solutions in nematic liquid-crystalline solvents.

Partially averaged dipolar couplings (also referred to as residual dipolar couplings) D(ij) can be obtained from the analysis of the NMR spectra of molecules dissolved in liquid-crystalline solvents. Their values for a nonrigid molecule depend upon the bond lengths and angles, the rotational potentials, and the orientational order of the molecules. The molecule studied, 1-chloro-2-bromoethane, is one of the simplest example of a substituted alkane in which the rotational potential has three minimum-energy positions, trans and gauche+/-conformations, and the present investigation explores the problems inherent in deriving the form of the potential and the molecular geometry from the set of partially averaged couplings between the protons, and between protons and (13)C nuclei. The geometrical parameters and the rotational potential obtained are compared with the results from a density-functional theory method.

Selective detection of the proton NMR spectra of molecules containing rare spins at natural abundance in liquid crystalline samples.

It is shown that the proton NMR spectra of molecules containing rare spins at natural abundance dissolved in a liquid crystalline solvent can be obtained free from the strong lines from the spectrum of the abundant isotopomer by the 2D HSQC NMR experiment. The technique can also give the individual chemical shifts of the rare spins, and, for a molecule containing another abundant nucleus, such as fluorine, the rare spin--(19)F total anisotropic couplings are also obtained. The usefulness of the technique is demonstrated for molecules containing (13)C as the rare spins.

NMR experimental evidence of the differentiation of enantiotopic directions in C(s) and C(2v) molecules using partially oriented, chiral media.

We report new and explicit experimental evidence of the differentiation of (1)H-(1)H, (13)C-(1)H, and (13)C-(2)H enantiotopic directions in prochiral molecules with C(s) and C(2v)symmetry dissolved in a chiral liquid-crystalline phase using (13)C and (2)H-[(1)H] NMR spectroscopy at the natural abundance level. The case of endo-bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, bicyclo[2.2.1]hepta-2,5-diene, and ethyl alcohol oriented in organic solutions of poly-gamma-benzyl-L-glutamate (PBLG) or poly-epsilon-carbobenzyloxy-L-lysine (PCBLL) is investigated and discussed. Next, we describe the first NMR differentiation of enantiotopic directions in a C(2v) molecule with no prostereogenic carbon using malononitrile as a model. The various results presented in this work experimentally validate our recent theoretical arguments which predict that NMR spectra of nonplanar C(s) and C(2v) compounds embedded in a chiral oriented solvent should differ from those recorded in nonchiral oriented media, because their effective molecular symmetry point group (interacting molecule) is different from their molecular point group (isolated molecule). In addition, the differentiation of enantiotopic directions in C(2v) molecules exhibiting no prostereogenic tetrahedral center illustrates for the first time an old stereochemical hypothesis which speculates that "for molecules of the type CXXYY. the two X groups as well as the Y groups are equivalent and cannot be distinguished in chiral or achiral circumstances. However, the relationships between X and Y groups are not all equivalent. The four X-Y relationships may be ordered into two enantiotopic sets of two equivalent relationships" (Mislow, K.; Raban, M. Top. Stereochem. 1967, 1, 1) and validate the stereogenicity concepts proposed more recently by Fujita (Fujita, S. J. Am. Chem. Soc. 1990, 112, 3390).

Soil solutions and surface water analysis in two contrasted watersheds impacted by acid deposition, Vosges mountains, N.E. France: interpretation in terms of Al impact and nutrient imbalance.

Two acid watersheds in the Vosges mountains (N.E. France), one with a podzol, the other with an acid brown soil, were monitored by analysing soil solutions and two related springs for total chemistry and Al speciation. High concentrations of inorganic Al did not occur in the podzol upper horizons but were evident in the corresponding spring, together with low concentrations of Ca. In the acid brown soil area, high concentrations of inorganic Al occurred in the leaching water draining the upper soil horizons, but not in the undrained water nor in the spring, the latter exhibiting rather high Ca content. In both watersheds, needle yellowing in conifers could be observed and might be ascribed to Mg deficiency rather than to Al toxicity.

[Methods of in vitro evaluation for nephrotoxicity]. / Les méthodes d'évaluation in vitro de la néphrotoxicité.

Mechanisms and precise targets of nephrotoxicants have been evidenced in experimental animals, a very important contribution for decades. But, structural and functional heterogeneity in renal tissue makes often difficult the exact determination of the molecular targets at renal cell level. Indeed, some new in vitro models have been gradually reported. This non exhaustive review purposes to present the different recent models, ever more reductionist, as isolated kidneys, kidney slices, isolated renal cells or renal cultured cells to assess the nephrotoxicity of new molecules. For every model are presented a brief history, the principle of the technique, viability criteria and its relative interest for renal toxicology. In conclusion, a general discussion points out the main advantages and limits of each one.

[Isolation of tubular proximal cells of the human kidney by affinity chromatography using Lotus tetragonolobus lectin]. / Isolement de cellules tubulaires proximales de reins humains par chromatographie d'affinité grâce à la lectine de Lotus tetragonolobus.

An affinity chromatography technique, using a lectin issued from Lotus tetragonolobus as ligand, and coupled to Sepharose 6MB support, has been chosen to enrich a cellular population in tubular proximal cells. Cell desorption was obtained with a competitive sugar, L Fucose. Intensive enzymatic brush border activity, shown in selected cells compared with initial cellular mixture, evidenced an enrichment in proximal tubular cells. These results prove that lectin use as ligand in affinity chromatography permits the separation of renal cell populations and presents the advantage that the bound cells can be recovered by a gentle competitive elution for further cultures.

[Effect of cisplatin on healthy human renal tubular cells and cancerous cells in culture]. / Effet du cis-platine sur des cellules tubulaires rénales humaines saines et cancéreuses en culture.

An in vitro comparative cis-platin cytotoxicity was evidenced in human renal normal and tumoral cultured cells. Cytotoxic effect was estimated at membrane level by intracellular enzymatic markers release into culture medium and nuclear level by ADN synthesis inhibition after 3H thymidine incorporation. These results show a more intensive activity of cis-platin in normal tubular cells than in tumoral ones.

[Morphologic and enzymatic characteristics of healthy human kidney epithelial tubular cells in culture]. / Caractéristiques morphologiques et enzymatiques de cellules épithéliales tubulaires de rein humain sain en culture.

The present study defines morphological and enzymatic characteristics of human healthy renal tubular cells in primary culture, and during three passages. The results confirm the ability to culture human renal tubular cells and to quantitatively evaluate the activity of specific tubular enzymes in the cells and in the culture medium. Such determinations can be compared to similar enzyme activity research in renal biopsies or in urines, to investigate human renal cell functions, in clinical, pharmacological or toxicological approaches.

An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release.

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl(2) concentrations (5, 10, 20, and 50 µg). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 µg and 10 µg HgCl(2)/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 µg/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.

New insecticidal cyclodepsipeptides from the fungus Isaria felina. I. Production, isolation and insecticidal properties of isariins B, C and D.

Three new cyclodepsipeptides related to the previously described isariin were isolated from a strain of Isaria felina. They were named isariins B, C and D. Isariin D and, to a lesser extent, isariin C, exhibited insecticidal activity against Galleria mellonella larvae, whereas isariin B and isariin itself proved inactive.