Jean-Yves Lallemand

Ether linkage between phenolic acids and lignin fractions from wheat straw

Abstract The bonding of the bound-phenolic acids present in three lignin preparations isolated from wheat straw where determined. p -Coumaric acid was mainly ester-linked whereas 35–75% of the recovered ferulic acid was ether-linked to milled straw lignin or enzyme lignin. Ferulic acid ethers accounted for 1.1% dry wt of alkali extracted lignin and might explain the high solubility of Gramineae lignins in soda. Isolated lignins were associated to hemicelluloses, principally arabinoglucuronoxylans. The possible existence of ferulic acid cross-links between lignin and arabinoglucuronoxylans is discussed.

Comparison of Wheat Straw Lignin Preparations - I. Chemical and Spectroscopic Characterizations

Alkali lignin was isolated from wheat straw and compared to two lignin preparations isolated from the same sample by a procedure which allowed to show an heterogeneity of rnonomeric composition between lignin fractions isolated from poplar wood. Using C NMR-, UV-, IRspectroscopy and nitrobenzene oxidation, no significative difference in monomeric composition was found in the case of wheat; difference of condensation degree between these fractions was however suggested by nitrobenzene oxidation. Arabinoxylans, proteins and silica were found in each preparation and a very close association between lignin and polysaccharides is suggested.

Photosynthetically 13C-Labelled Poplar Lignins: - 13C NMR Experiments

Poplars were grown from cuttings under air containing CO2 with C at about 20% C, in a growth chamber during three months. C-labelled lignin fractions were isolated and characterized by C NMR spectroscopy. Advantage was taken of the C enrichment to perform C NMR resolution enhancement and editing experiments. Some effects of LiCl, added to the DMSO-d6 Solutions, on the C NMR spectra are discussed. It was also attempted to assign more accurately the polysaccharidic peaks found in the C NMR spectra of lignin fractions in Order to study the lignin-carbohydrate relationships.

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2☆

Abstract The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and cis -[Pt(NH3)2(H2O)2](NO3)2 (8.4 × 10−6 to 1.3 × 10−4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent 1H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the cis -PtII(NH3)2 moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10−3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.

1H NMR conformational study of sulfated and non-sulfated cholecystokinin fragment CCK27–33: Influence of the sulfate group on the peptide folding

1H NMR study of cholecystokinin fragment (CCK27-33) in (C2H3)2SO and in 2H2O at different pH shows that sulfated (CCK7) and non sulfated (NS-CCK7) peptides are under preferentially folded conformations characterized by a beta-turn including the sequence Gly-Trp-Met-Asp with a H-bond between the CO of Gly and the NH of Asp. This structure is probably stabilized by an ionic interaction between Tyr and Asp. Moreover, the N-terminal part of CCK7 forms a C7 structure with a weak H-bond between the CO of Gly and the NH of Trp. In this model all CCK7 hydrophobic side chains are in close vicinity, far from the hydrophilic sulfate group. Full interaction with brain CCK8 receptors could require both the sulfate group and the maintaining of conformational constraints.

PARIS, a program for automatic recognition and integration of 2D NMR signals

The quantitative evaluation of 2D maps is the requisite step for some of the most fascinating recent developments in NMR techniques such as, for example, the use of the full relaxation matrix over a series of 2D NMR maps corresponding to various mixing times leading to a more accurate description of 3D geometries of macromolecules ( I, 2)) and the analysis of the cross-peak fine structure based on either pattern recognition (3,4) or symmetry reduction (57) algorithms allowing a more extensive description of the coupling networks. However, due to the increasing complexity of the systems and particularly the growing number of proteins and nucleic acid fragments (Fig. 1) under study in many laboratories, manual extraction of the information present in the 2D NMR spectra, but dispersed in hundreds of peaks, is extremely tedious if not impossible. Computeraided approaches are therefore essential in any practical solution. Moreover, an automated 2D spectra analysis should be easily implemented on commercial computers, have a fast algorithm, even on the smaller computers normally used for controlling the spectrometer operations, and be reasonably efficient when operating under the difficult conditions usually encountered in the study of biological macromolecules (low signal-to-noise ratio, presence of overlapping peaks, and presence of artifacts). The automated recognition methods of Madi et al. (6) and of Glaser and Kalbitzer ( 7) are presently to our knowledge the only fully automated approaches for 2D spectra reported in the literature, besides Pfandler’s pattern recognition technique (3, 4) which is of a more global philosophy. Another very interesting possibility (8) relies on the prior knowledge of those individual 1 D signals which could give rise to cross peaks. This facilitates the task but is inherently restricted in scope, particularly for larger spin systems. The methodology we propose here is based on the identification of the local extrema, followed by a precise evaluation of the peak extent. Its main disparity with the preceding ones lies in its completely different approach for the peak extent definition module, which allows a much clearer separation of the cross peaks in crowded regions. Moreover, since in many applications such as NOESY and HOHAHA spectra the fine structure present in the cross peaks merely complicates the analysis, we devised an algorithm that is able, at will, to handle cross peaks with or without fine structure. The program may roughly be broken into two consecutive operations, namely the extraction of the local maxima (peak picking) and the determination of the in-

ATP Binding Cassette transporters associated with chemoresistance: transcriptional profiling in extreme cohorts and their prognostic impact in a cohort of 281 acute myeloid leukemia patients

Background A major issue in the treatment of acute myeloid leukemia is resistance to chemotherapeutic drugs. An increasing number of ATP-Binding-Cassette transporters have been demonstrated to cause resistance to cancer drugs. The aim of this study was to highlight the putative role of other ATP-Binding-Cassette transporters in primary chemoresistant acute myeloid leukemia. Design and Methods In the first part of this study, using taqman custom arrays, we analyzed the relative expression levels of 49 ATP-Binding-Cassette genes in 51 patients divided into two extreme cohorts, one very sensitive and one very resistant to chemotherapy. In the second part of this study, we evaluated the prognostic impact, in a cohort of 281 patients, of ATP-Binding-Cassette genes selected in the first part of the study. Results In the first part of the study, six genes (ATP-Binding-CassetteA2, ATP-Binding-CassetteB1, ATP-Binding-CassetteB6, ATP-Binding-CassettC13, ATP-Binding-CassetteG1, and ATP-Binding-CassetteG2) were significantly over-expressed in the resistant group compared with the sensitive group. In the second cohort, overexpression of 5 of these 6 ATP-Binding-Cassette genes was correlated with outcome in univariate analysis, and only the well-known ATP-Binding-CassetteB1 and G2, and the new ATP-Binding-CassetteG1 in multivariate analysis. Prognosis decreased remarkably with the number of these over-expressed ABC genes. Complete remission was achieved in 71%, 59%, 54%, and 0%, (P=0.0011) and resistance disease in 21%, 37%, 43%, and 100% (P<0.0001) of patients over-expressing 0, 1, 2, or 3, ABC genes, respectively. The number of ATP-Binding-Cassette genes expressed, among ATP-Binding-CassetteB1, G1, and G2, was the strongest prognostic factor correlated, in multivariate analysis, with achievement of complete remission (P=0.01), resistant disease (P=0.01), and overall survival (P=0.02). Conclusions Using expression profiling, we have emphasized the diversity of ATP-Binding-Cassette transporters that cooperate to promote chemoresistance rather than overexpression of single transporters and the putative role of new ATP-Binding-Cassette tranporters, such as ATP-Binding-CassetteG1. Modulation of these multiple transporters might be required to eradicate leukemic cells.

Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642-Ser871) and NBD2 (Ser1286-Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10(-5) s(-1)) and NBD2 (6x10(-6) s(-1)) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 microM. Despite the fact that both monomers bind ATP, no stable NBD1.NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1.NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1.NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1.NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar coupling and small angle X-ray scattering data

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the γS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.

Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary1H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel β-sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu37, Leu23, Val47, Tyr14, Trp45 and Tyr5. The Trp45 and Tyr5 rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.