Untargeted metabolomics approach to discriminate mistletoe commercial products.

Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe's anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure-activity relationships based on LC-MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Does the Phytochemical Diversity of Wild Plants Like the Erythrophleum genus Correlate with Geographical Origin?

Secondary metabolites are essential for plant survival and reproduction. Wild undomesticated and tropical plants are expected to harbor highly diverse metabolomes. We investigated the metabolomic diversity of two morphologically similar trees of tropical Africa, Erythrophleum suaveolens and E. ivorense, known for particular secondary metabolites named the cassaine-type diterpenoids. To assess how the metabolome varies between and within species, we sampled leaves from individuals of different geographic origins but grown from seeds in a common garden in Cameroon. Metabolites were analyzed using reversed phase LC-HRMS(/MS). Data were interpreted by untargeted metabolomics and molecular networks based on MS/MS data. Multivariate analyses enabled us to cluster samples based on species but also on geographic origins. We identified the structures of 28 cassaine-type diterpenoids among which 19 were new, 10 were largely specific to E. ivorense and five to E. suaveolens. Our results showed that the metabolome allows an unequivocal distinction of morphologically-close species, suggesting the potential of metabolite fingerprinting for these species. Plant geographic origin had a significant influence on relative concentrations of metabolites with variations up to eight (suaveolens) and 30 times (ivorense) between origins of the same species. This shows that the metabolome is strongly influenced by the geographical origin of plants (i.e., genetic factors).

Optimization of Experimental Parameters to Explore Small-Ligand/Aptamer Interactions through Use of (1) H†NMR Spectroscopy and Molecular Modeling.

Aptamers constitute an emerging class of molecules designed and selected to recognize any given target that ranges from small compounds to large biomolecules, and even cells. However, the underlying physicochemical principles that govern the ligand-binding process still have to be clarified. A major issue when dealing with short oligonucleotides is their intrinsic flexibility that renders their active conformation highly sensitive to experimental conditions. To overcome this problem and determine the best experimental parameters, an approach based on the design-of-experiments methodology has been developed. Here, the focus is on DNA aptamers that possess high specificity and affinity for small molecules, L-tyrosinamide, and adenosine monophosphate. Factors such as buffer, pH value, ionic strength, Mg(2+) -ion concentration, and ligand/aptamer ratio have been considered to find the optimal experimental conditions. It was then possible to gain new insight into the conformational features of the two ligands by using ligand-observed NMR spectroscopic techniques and molecular mechanics.

Use of a fractional factorial design to study the effects of experimental factors on the chitin deacetylation.

Chitosan is obtained by deacetylation of chitin. Chitosan versatility is directly related to the polymer's characteristics depending on the deacetylation process. The aim of this research was to study the parameters influencing deacetylation and to elucidate their effect on acetylation degree (DA) and molecular weight (MW). The effect on chitosan DA was investigated using a fractional factorial design 2(7-3) with seven factors and two variation levels. The tested factors were: X1=number of successive baths, X2=reaction time, X3=temperature, X4=alkali reagent, X5=sodium borohydride, X6=the atmospheric conditions and X7=alkali concentration. A mathematical model was investigated corresponding to the following relation y=7.469-1.344X1-1.094X2-3.094X3+1.906X4+0.656X5+0.906X6-1.031X7+0.469X1X2-0.781X3X4+0.906X1X3X4 with R2=0.99. This model allows fixing experimental conditions for each desired DA. To study the effect on chitosan MW, only atmospheric conditions and use of sodium borohydride as an oxygen scavenger were investigated. The use of sodium borohydride and nitrogen atmosphere was found to have a protective effect against chitosan degradation during deacetylation.

Physicochemical characterization of α-, ß-, and γ-cyclodextrins bioesterified with decanoate chains used as building blocks of colloidal nanoparticles.

Nanoparticles of amphiphilic α-, ß-, and γ-cyclodextrins were obtained by formulation of cyclodextrins enzymatically transesterified with vinyl decanoate. The product of this synthesis is a mixture of bioesterified cyclodextrins with various degrees of substitution (DS) presenting for a same DS different regio-isomers. In a first step, the efficiency of a MALDI-TOF procedure to characterize the average molecular weight of the derivative bulk mixture was demonstrated by comparing the results with those obtained from complementary NMR and HPLC techniques. In a second step, the ultrastructure of nanoparticles prepared from three different batches of synthesis was investigated and correlated with the average molecular weight and DS of the parent derivative.

Enantioseparation by MEKC using a ligand exchange-based chiral pseudostationary phase.

In this paper, a new ligand-exchange -MEKC mode, based on the design of a unique lipohilic species (4'-octadecylneamine derivative), which served both as micelle-forming surfactant (by its hydrophobic part) and central ion-complexing ligand (by its hydrophilic part) is described. The CMC of the used lipophilic neamine derivative was first determined by surface tension measurements. Subsequent NMR experiments were performed in order to investigate the Cu(II) binding properties of the neamine micellar phase. The enantioseparation properties of both the octadecylneamine derivative-Cu(II) MEKC and the native neamine-Cu(II) CE systems were evaluated and compared using the tryptophan racemate as a probe analyte. The effects of several different electrophoretic conditions on the enantiomer migration behavior in the ligand-exchange-MEKC mode were examined. The developed methodology was also applied to the enantioseparation of other analytes such as 1-methyl-tryptophan, 3,5-diiodo-tyrosine and 1-naphtyl-alanine.

NMR characterization of the interaction between the C-terminal domain of interferon-gamma and heparin-derived oligosaccharides.

Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNgamma (interferon-gamma) is secreted by activated T-cells and natural killer cells. IFNgamma is involved in a wide range of physiological processes, including antiviral activity, immune response, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFNgamma activity is modulated by the binding of its C-terminal domain to HS (heparan sulphate), a glycosaminoglycan found in the extracellular matrix and at the cell surface. In the present study, we analysed the interaction of isolated heparin-derived oligosaccharides with the C-terminal peptide of IFNgamma by NMR, in aqueous solution. We observed marked changes in the chemical shifts of both peptide and oligosaccharide compared with the free state. Our results provide evidence of a binding through electrostatic interactions between the charged side chains of the protein and the sulphate groups of heparin that does not induce specific conformation of the C-terminal part of IFNgamma. Our data also indicate that an oligosaccharide size of at least eight residues displays the most efficient binding.

Unusual Ca(2+)-calmodulin binding interactions of the microtubule-associated protein F-STOP.

F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain. We present here an analysis of this CaM binding interaction and find that it is highly unusual in nature. For this work, we synthesized two model peptides of a single STOP central repeat motif and analyzed their binding to CaM by fluorescence, circular dichroism, infrared and NMR spectroscopy. Both peptides bind to CaM with an affinity of 4 microM, similar to that of the native protein. Results indicate that the peptides bind CaM in an atypical manner. Binding is highly dependent on the concentration of cations, indicating that it is to some extent electrostatic. Further, IR and CD analysis shows that, in contrast to typical CaM binding reactions, CaM does not change in helical structure on binding. NMR mapping confirms that CaM remains in extended conformation on binding a single STOP peptide. Binding of a single peptide to CaM occurs principally in the CaM C-terminal region, and the C-terminal domain of CaM effectively competes for STOP binding. Our results establish that CaM binds STOP in an unusual manner, involving mainly the C-terminus of CaM, thus leaving CaM potentially accessible for another binding partner at the N-terminus. This intriguing possibility could be of physiological importance in F-STOP mediated CaM regulation of microtubule dynamics or stability, specifically during mitosis where CaM and STOP colocalize.

Conformational analysis of the exopolysaccharide from Burkholderia caribensis strain MWAP71: impact on the interaction with soils.

The strain MWAP71 of Burkholderia caribensis produces a branched charged exopolysaccharide (EPS) that is responsible for soil aggregation. Understanding the conformational properties of the isolated polysaccharide is a prerequisite for proper investigation of the interactions between the polysaccharide and the soil at the atomic level. The aim of this study is first to have an overall view of the flexibility of the backbone and then to ascertain the role played by side groups in the conformational properties of the main chain. Conformational analysis of each oligomeric segment of the polysaccharide has been performed by means of adiabatic mapping of the backbone glycosidic torsion angles using the MM3(92) force field. Substitution by an acetyl group or by a Kdo unit has only a slight effect on the potential energy surfaces of the fragment model compounds. Calculated partition functions, however, indicate that the overall flexibility is slightly larger for the substituted oligomers than for the unsubstituted ones. Prediction of selected average interproton distances from the AB and BC potential energy surfaces allows comparison between modeling results and NMR measurements performed on the ABC fragment. Agreement between the experimental and the predicted data suggests that the established surfaces correctly reflect the observed conformational behavior of such fragments and validate the modeling protocol. The above results have been extended to regular and disordered long polymer chains, differing in Kdo content. It is found that Kdo affects the helical conformations of the polysaccharide. The number of stable helices is considerably larger with Kdo than without Kdo. On the contrary, Kdo has only a moderate effect on unperturbed disordered conformations of the polysaccharide. Predicted persistence length of 70 A suggests that the polymer is semirigid with moderate extension. A further validation of the modeling results is obtained by the good concordance between this predicted value and the experimental one of 95 A, measured from light scattering and viscosity experiments. The results lead to an understanding of the interactions of this polysaccharide with soils.