In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos.

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.

How to build an adapted and bioactive cell microenvironment? A chemical interaction study of the structure of Ca-alginate matrices and their repercussion on confined cells.

Alginates are increasingly being used as medical materials (matrices for tissue regeneration, surgical sponges, hemostatic bandages, microbial and cell encapsulation, artificial bacterial biofilms, etc.). The constitution of alginate gel networks is a complex phenomenon. A great number of different kinds of polysaccharidic frameworks can come to existence depending on the conditions used for their attainment. For instance, the degree of heterogeneity and porosity of Ca-alginate beads rely on this molecular organization. The formation of structural irregularities (superficial crust, cavities, shafts, dense or light gel frameworks, ordered or chaotic domains, etc.) within the alginate gel beads are inherent to this skeletal design. Several specific staining molecules (e.g. calcon carboxylic acid, murexide, methylene blue) that are negatively or positively charged interact with the gel network. These molecules allowed us to reveal a great variety of chemical interactions shown by the pattern coloration of the internal structure of the gel. The results observed are very different for the several matrices analyzed, which could explain to a great extent the singular behavior that cells confined in these kind of matrices exhibit.

Kinetic study of littorine rearrangement in Datura innoxia hairy roots by (13)C NMR spectroscopy.

The kinetics of tropane alkaloid biosynthesis, particularly the isomerization of littorine into hyoscyamine, were studied by analyzing the kinetics of carbon-13 ((13)C) in metabolites of Datura innoxia hairy root cultures fed with labeled tropoyl moiety precursors. Both littorine and hyoscyamine were the major alkaloids accumulated, while scopolamine was never detected. Feeding root cultures with (RS)-phenyl[1,3-(13)C(2)]lactic acid led to (13)C spin-spin coupling detected on C-1' and C-2' of the hyoscyamine skeleton, which validated the intramolecular rearrangement of littorine into hyoscyamine. Label from phenyl[1-(13)C]alanine or (RS)-phenyl[1,3-(13)C(2)]lactic acid was incorporated at higher levels in littorine than in hyoscyamine. Initially, the apparent hyoscyamine biosynthesized rate (v(app)()hyo = 0.9 micromol (13)C.flask(-1).d(-1)) was lower than littorine formation (v(app)()litto = 1.8 micromol (13)C.flask(-1).d(-1)), suggesting that the isomerization reaction could be rate limiting. The results obtained for the kinetics of littorine biosynthesis were in agreement with the role of this compound as a direct precursor of hyoscyamine biosynthesis.

Studies of low molecular weight samples of glucuronans with various acetylation degree.

Partially acetylated, high molecular weight glucuronans were produced by a Sinorhizobium meliloti mutant strain. Two native glucuronan samples with various degrees of acetylation were sonicated to obtain lower molecular weight samples and with low viscosity suitable for chemical modification and (13)C NMR experiments. The average degree of substitution (DS) of the polymer was estimated by Fourier transform infrared (FTIR) and NMR. (13)C NMR spectra were obtained and used to suggest a complete assignment of the signals. The nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multi-bond coherence (HMBC) experiments were used to elucidate connectivities between the various residues and deduce the linkage of these residues within the polysaccharide.