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Rudd T.R.,Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni | Rudd T.R.,University of Liverpool | Yates E.A.,University of Liverpool
Molecular BioSystems | Year: 2012

The form of the biosynthetic pathway of the biologically and medically important polysaccharides heparan sulfate (HS) and the closely related heparin remain obscure despite significant progress characterising the biosynthetic machinery. Considering possible biosynthetic schemes using a graph approach and applying known constraints of enzyme order and specificity, a previously unreported system with a highly efficient tree structure emerged with two features: (1) All commonly occurring HS disaccharides could be synthesised through a common route, the major branch. (2) The least common disaccharides also occurred on a separate common branch, termed here the minor branch. This suggested that the relative abundance of these two sets of structures were the result of the specificity of a single enzyme (HS epimerase) acting at an early point in the scheme, to convert GlcA-GlcNS to IdoA-GlcNS in preference to converting GlcA-GlcNAc to IdoA-GlcNAc. A third key finding was that the common substrates for 3-O-sulfation all lie on the same (major) branch. The proposed scheme is consistent with a wide body of experiments comprising both biochemical data and results from HS biosynthetic enzyme knockout experiments in the literature. The major branch also contains a bifurcation, providing a choice of two distinct backbone geometries with the same charge. Further development of this novel biosynthetic scheme, in which frame shifts in the site of action of the enzymes were permitted to occur, while maintaining their order of action, suggested a mechanism by which domains could be generated, or further modification blocked. The relationship between the proposed pathway and the geometric and charge possibilities it allows were also explored. © 2012 The Royal Society of Chemistry. Source

Pezzoli D.,Polytechnic of Milan | Olimpieri F.,Polytechnic of Milan | Malloggi C.,Polytechnic of Milan | Bertini S.,Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni | And 2 more authors.
PLoS ONE | Year: 2012

Background: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI x) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. Methodology/Principal Findings: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI x derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI x series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI x copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI 2.7% was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. Conclusions/Significance: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI x copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity. © 2012 Pezzoli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

Sarrazin S.,CNRS Institute of Pharmacology and Structural Biology | Sarrazin S.,University of California at San Diego | Lyon M.,University of Manchester | Deakin J.A.,University of Manchester | And 4 more authors.
Glycobiology | Year: 2010

Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40 kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and are mostly clustered in tetra- (35%), hexa- (12%), and octa- (5%) saccharide domains. Highly sulfated ΔD, ΔE, and ΔB disaccharide units (ΔHexA2S-GalNAc6S, ΔHexA-GalNAc4S6S, and ΔHexA2S-GalNAc4S) were also detected in significant amounts in both chains and may account for the HGF/SF-binding activity of the CS/DS. This work establishes that HEK-293 cells can be engineered to provide a valuable source of Endocan with authentic CS/DS chains, enabling the purification of sufficient amounts for structural and/or binding analysis and providing a possible model of Endocan CS/DS chain organization. © The Author 2010. Published by Oxford University Press. All rights reserved. Source

Piazza L.,University of Milan | Bertini S.,Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni | Milany J.,Sari University of Agricultural Sciences and Natural Resources
Carbohydrate Polymers | Year: 2010

In this work a physico-chemical characterization of the polysaccharide fraction of the exudates of Launaea acanthodes, a common medicinal species in central regions of Iran, was performed. The extraction of the polysaccharide fraction of the exudates was described. The structure characterization of the polysaccharide was performed with mono and bi-dimensional NMR spectroscopy techniques, which allowed the chemical composition and the relative monomers abundance of the extracted fraction to be estimated. The constituent monosaccharides were predominantly galactose, rhamnose, arabinose and galacturonic acid residues. Interchain association phenomena in the main arabinogalactans component (Mw = 33.550 Da) were evidenced both from high-performance size-exclusion chromatography (HP-SEC-TDA), and by intrinsic viscosity ([η]0 = 0.323 dl g-1 in water and [η]0 = 0.115 dl g-1 in NaNO3 0.1 M at 20 °C) measurement and the Huggings coefficient λ. Due to the formation of hyperentanglements, a unique function of C[η]0 cannot be defined. The technological consequence was that once structured, the polysaccharide network did not express a sufficient degree in polymer orientation and therefore cast films showed weak mechanical properties. © 2009 Elsevier Ltd. All rights reserved. Source

Scalenghe R.,University of Palermo | Minoja A.P.,Bruker | Zimmermann S.,Swiss Federal Institute of forest | Bertini S.,Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni
Geoderma | Year: 2016

In forests, soils contain at least twice as much carbon than plants that mostly grow in the upper layers. Litter at the interface between soils and the atmosphere regulates a variety of biogeochemical cycles, which are important for both plants and soils and have possible implications for other environmental components. We have compared leachates collected during an incubation experiment on: a) two deciduous leaves; b) organic and mineral horizons; c) treated with litter removal (and untreated) plots, to assess the changes in the chemical composition of the litter layers and leachates during weathering and their influence on the underlying horizons. Two different types of broadleaves - beech and oak - become indistinguishable when they experience weathering. As a litter horizon is altered, it becomes more stable and loses fewer elements, both in gaseous and liquid forms. The annual removal of litter represents a net loss of biomass from the system. Nevertheless, the effect on soil in the medium term is not significant. Leaves and litter horizons were incubated in micro-lysimeters, leached, and characterised by different analytical approaches, from elementary analyses (dissolved organic carbon, CO2 production, nitrogen forms, UV absorptivity) to solid state NMR spectroscopy. The results reveal that the removal of the litter does not degrade the underlying soils, in direct contrast to what was thought to be the case previously. Moreover, it extends previous knowledge that litter removal promotes an increase in fulvic acid activity in underlying horizons. The results demonstrate how this human disturbance, if not combined with other degradation factors, could promote podzolisation. In a wider outlook, if managed properly (for example, by burying litter removed after its use in animal husbandry), even the repeated removal of forest biomass contribute not negatively to the genesis of these soils. © 2016 Elsevier B.V.. Source

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