Potential use of biochar, compost and iron grit associated with Trifolium repens to stabilize Pb and As on a multi-contaminated technosol.

Vegetation cover can be used in the phytomanagement of polluted areas by adding value to abandoned sites and reducing the dispersion of pollutants by erosion. Appropriate amendments, that allow both efficient plant growth and the immobilization of contaminants in the soil must be chosen in order to optimize the efficiency of this process. We used a mining technosol mainly contaminated by arsenic (1068 mg kg-1) and lead (23387 mg kg-1) to study the effect of three amendments (biochar, compost and iron grit) on (i) physico-chemical properties of the soil and soil pore water, (ii) metal(loid) mobility, bioavailability and bioaccessibility (CaCl2 and Simple Bioaccessibility Extraction Test (SBET)), and (iii) the capability of Trifolium repens to germinate and grow. All the amendments used increased the pH and electrical conductivity of the SPW, resulting in a 90% decrease in the concentration of lead in the soil pore water (SPW). We also demonstrated a decrease in Pb phytoavailability. The amendments allowed the establishment of a plant cover, although the addition of iron grit alone did not allow any clover germination. For the Pontgibaud technosol, the combination of the three amendments resulted in a significant decrease in As and Pb concentrations in clover tissues, mainly in the aerial organs. The amendments also made it possible for some of them to halve the phytoavailable fraction of arsenic. However, for compost, both the As concentrations in the SPW, and the bioavailable fraction of As increased. All the amendments used had contrasting effects on the bioaccessible fractions of metal(loid)s. The most efficient amendment combination was the addition of 5% biochar and 5% compost.

Capability of amendments (biochar, compost and garden soil) added to a mining technosol contaminated by Pb and As to allow poplar seed (Populus nigra L.) germination.

The germination capacity of poplar seeds has never been studied in the context of metal(loid)-contaminated soils, even though poplars are present over a vast geographical area. In this study, black poplar seeds from the Loire Valley (France) were grown for 28 days in mesocosm on a heavily polluted soil that was subjected to different amendments. This phytomanagement process aimed to allow the revegetation of an As and Pb-contaminated mining soil by adding appropriate amendments, resulting in metal(loid) soil stabilisation and efficient plant growth. The objectives were to evaluate the effect of three amendments (garden soil, compost and biochar) when added alone or combined to a technosol on (i) the soil physicochemical properties, (ii) the mobility of As and Pb in the soil pore water (SPW), (iii) the capacity of poplar seeds to germinate and to grow and (iv) the metal(loid) distribution within the plant organs. The addition of amendments alone or combined allowed a 90% decrease in SPW Pb concentrations, while the arsenic concentrations were between 18 and 416 times higher. However, we were only able to obtain seed germination and plant growth on amended soils. These promising results will allow us to explore the use of such amendments in rehabilitating areas that are sources of significant metal(loid) dissemination, as well as allowing a natural plant recolonisation of these sites by seeds from the surrounding environment.

Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation.

A novel category of major intrinsic proteins which share weak similarities with previously identified aquaporin subfamilies was recently identified in land plants, and named X (for unrecognized) intrinsic proteins (XIPs). Because XIPs are still ranked as uncharacterized proteins, their further molecular characterization is required. Herein, a systematic fine-scale analysis of XIP sequences found in flowering plant databases revealed that XIPs are found in at least five groups. The phylogenetic relationship of these five groups with the phylogenetic organization of angiosperms revealed an original pattern of evolution for the XIP subfamily through distinct angiosperm taxon-specific clades. Of all flowering plant having XIPs, the genus Populus encompasses the broadest panel and the highest polymorphism of XIP isoforms, with nine PtXIP sequences distributed within three XIP groups. Comprehensive PtXIP gene expression patterns showed that only two isoforms (PtXIP2;1 and PtXIP3;2) were transcribed in vegetative tissues. However, their patterns are contrasted, PtXIP2;1 was ubiquitously accumulated whereas PtXIP3;2 was predominantly detected in wood and to a lesser extent in roots. Furthermore, only PtXIP2;1 exhibited a differential expression in leaves and stems of drought-, salicylic acid-, or wounding-challenged plants. Unexpectedly, the PtXIPs displayed different abilities to alter water transport upon expression in Xenopus laevis oocytes. PtXIP2;1 and PtXIP3;3 transported water while other PtXIPs did not.

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group.

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphor-othioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/micromol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphor-amidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.

Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification.

A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6'-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated thiol group is immediately protected by action of the second reagent. The optimal conditions were determined by taking into account the stability and the reactivity of both reagents with regards to pH and temperature. This method was validated through two applications, the substitution of bovine serum albumin with a bromoacetyl peptide and the substitution of an amino link at the 5' end of an oligonucleotide by reaction with either a fluorescent tag, iodoacetamidofluorescein, or a bromoacetyl peptide, upon reduction of the protected disulfide bridge with a third water-soluble reagent, namely tris(2-carboxyethyl)phosphine.

Glycotargeting: the preparation of glyco-amino acids and derivatives from unprotected reducing sugars.

Lectins are present on the surface of many cells. Many lectins actively recycle from membrane to endosomes and efficiently take up glycoconjugates in a sugar-dependent manner. On this basis, glycoconjugates, specially those obtained by chemical means, are good candidates as carriers of drugs, oligonucleotides or genes. In this paper, we present a panel of methods suitable to transform unprotected reducing oligosaccharides into glycosynthons designed to be easily linked to therapeutic agents. All the glycosynthons presented here are glycosylamines or derivatives, mainly glyco-amino acids or glycopeptides. Glycosylamines are easy to obtain, but they are very labile in slightly acidic or neutral medium; they must be stabilized, by acylation for instance. The coupling efficiency of a reducing sugar with ammonia as well as an alkylamine or an arylamine is higher at high temperature, however, because of the Amadori rearrangement, special conditions have to be selected to prepare the expected glycosylamine derivative with a high yield. Glycosylamines are easily acylated by N-protected amino acids, or by halogeno acids which can then be transformed into amino acids. Alternatively, unprotected reducing oligosaccharides may very efficiently be transformed into N-glycosyl-amino acids and then protected by N-acylation. With a glutamyl derivative having both the alpha-amino and the gamma-carboxylic groups free, the coupling and the acylation, which is intramolecular, are roughly quantitative. N-oligosaccharyl-amino acid derivatives are interesting glycosynthons, because their sugar moiety bears the specificity towards membrane lectins while the amino acid part has the capacity to easily substitute a therapeutic agent.

Novel glycosynthons for glycoconjugate preparation: oligosaccharylpyroglutamylanilide derivatives.

The reducing sugar of an oligosaccharide reacting with the alpha-amino group of an amino acid is converted to an N-oligosaccharylamino acid which can then be stabilized by N-acylation. Oligosaccharides in solution in N,N-dimethylformamide reacted with alpha-glutamyl-p-nitroanilide at 50 degrees C for a few hours, leading to an N-oligosaccharylglutamyl-p-nitroanilide. Then, the gamma-carboxylic group of the glutamyl moiety, activated by adding (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), reacted with the substituted alpha-amino group of the glutamyl residue, leading to an N-oligosaccharylpyroglutamyl-p-nitroanilide within 0.5 h. Such a one-pot two-step reaction was shown to be very efficient in the case of a disaccharide such as lactose, or pentasaccharides such as lacto-N-fucopentaoses, Lewis(a) or Lewis(x). The glycosynthons were characterized by chromatography (HPAEC and HPLC); their molecular mass was determined by electrospray ionization mass spectrometry, and the glycosylamides were shown to have a beta-anomeric configuration on the basis of their proton NMR. The N-oligosaccharylpyroglutamyl-p-nitroanilides are quite stable at room temperature over a large pH range. They are easily converted to N-oligosaccharylpyroglutamyl-p-isothiocyanatoanilides which can be used to prepare glycoconjugates such as cationic glycosylated polylysines suitable for specifically delivering genes or oligonucleotides in a sugar-dependent manner.

DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli.

The Escherichia coli melR gene encodes MelR protein which is a member of the AraC/XylS family of bacterial transcription activators. The function of MelR was investigated by making a targeted deletion in the melR gene of the Escherichia coli chromosome. MelR is a transcription activator essential for melibiose- dependent expression of the melAB operon which is needed for bacterial growth with melibiose as a carbon source. To investigate the interactions of MelR at the melAB promoter, both full length MelR and a shortened derivative, MelR173, containing the C-terminal DNA-binding domain, were purified as fusions to glutathione- S -transferase. Circular permutation studies show that both full-length MelR and MelR173 induce an apparent bend upon binding to target sites at the melAB promoter. Bound full-length MelR, but not MelR173, can oligomerise to form larger complexes that are likely to be involved in transcription activation.

Are there biological functions for bacterial endo-N-acetyl-beta-D-glucosaminidases?

The endo-N-acetyl-beta-D-glucosaminidases (ENGase) acting on the N-N'-diacetylchitobiosyl core of N-glycosylproteins are essential reagents for the investigation of the structure and the functions of glycoproteins. These enzymes were largely studied with the aim of offering more tools with new and broader substrate specificities to the community of glycobiologist. Conversely, little attention was given to their potential role in the physiology of bacteria, even though it had been shown that ENGases are important enzymes for the physiology of animal and plant cells. In this brief review, we present the main characteristics of the bacterial ENGases and confine our discussion to biological aspects of their action in bacterial systems.

Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin.

Known glycoproteins were used to determine the differences occurring in the binding specificities of the three variants of the K88 lectin in an approach essentially based on lectin blotting. During the screening, it was demonstrated that each variant of the K88 lectin biotinylated via its amino groups (NbioK88) exhibited a characteristic binding to the three chains of porcine fibrinogen. NbioK88ab weakly bound to A alpha chains, NbioK88ac bound to B beta and gamma chains, and NbioK88ad bound only to the gamma chain. To validate this model, the oligosaccharide moieties of porcine fibrinogen were analyzed with glycosidases and by lectin blotting and sugar composition. Both the B beta chain and gamma chain carry biantennary N-glycans of the N-acetyllactosamine type that are not recognized by K88 lectins. A alpha chains are substituted by sialylated T antigen. O-glycans were also detected on B beta and gamma chains of porcine fibrinogen and contribute to the recognition of these chains by K88ac and K88ad fimbriae.

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

Purification and characterization of an endo-N-acetyl-beta-D-glucosaminidase from the culture medium of Stigmatella aurantiaca DW4.

A novel endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans, was characterized in the culture medium of Stigmatella aurantiaca DW4. Purified to homogeneity by ammonium sulfate precipitation, gel filtration, and chromatofocusing, this ENGase presents, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a molecular mass near 27 kDa. Optimal pH and pI were 4.0 and 6.8, respectively. The enzyme, named ENGase St, exhibits high activity on oligomannoside-type glycoasparagines and glycoproteins and could also hydrolyze hybrid- and complex-type glycoasparagines but does not acts as a murein hydrolase.

A fluorescence high-performance liquid chromatography assay for enzymes acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans.

The glycoasparagine, Man7GlcNAc2Asn ('Man7') was labelled with resorufin and used as a specific substrate for the detection and quantification of endo-beta-N-acetyl glucosaminidases (Endos) acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans. Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidases (PNGases) cannot transform this substrate but they can be detected by the procedure described earlier using the resorufin-labelled N-glycopeptide [Glycoconjugate J., 9 (1992) 162-167]. These two substrates can be used in a simple, reproducible and very sensitive fluorescence HPLC assay in order to monitor Endo and PNGase activities during isolation and purification processes, or studies of the evolution of such activities during cultivation of the producing cells.

Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.

Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum.

Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.

Peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).