Sandbox article

doi:10.1016/j.carbpol.2017.07.073

Abstract

Silver linden flowers contain different pectins (PSI-PSIII) with immunomodulating properties. PSI is a low-esterified pectic polysaccharide with predominant homogalacturonan region, followed by rhamnogalacturonan I (RGI) with arabinogalactan II and RGII (traces) domains. PSII and PSIII are unusual glucuronidated RGI polymers. PSIII is a unique high molecular weight RGI, having almost completely O-3 glucuronidated GalA units with >30% O-3 acetylation at the Rha units. Linden pectins induced reactive oxygen species (ROS) and NO generation from non-stimulated whole blood phagocytes and macrophages, resp., but suppressed OZP-(opsonized zymosan particles)-activated ROS generation, LPS-induced iNOS expression and NO production. This dual mode of action suggests their anti-inflammatory activity, which is known for silver linden extracts. PSI expressed the highest complement fixation and macrophage-stimulating activities and was active on intestinal Peyer’s patch cells. PSIII was active on non-stimulated neutrophils, as it induced ß2-integrin expression, revealing that acetylated and highly glucuronidated RGI exhibits immunomodulating properties via phagocytes.

doi:10.1016/j.carbpol.2017.07.011

Abstract

Two pectic (chPS-L1, chPS-L2) and one polyphenolic (chPP-L) fractions were obtained from lavender flowers after boiling water extraction, exhaustive removing of alcohol-soluble molecules and SEC. chPS-L1 (52.4kDa) contains mainly low-acetylated and high-methoxylated homogalacturonans (HG), and smaller rhamnogalacturonan (RG) I backbone fragments rich in 1,3,5-branched arabinan and arabinogalactan (AG) II side chains. chPS-L2 (21.8kDa) contains predominantly similarly esterified HG, followed by RGI with AGII structures and RGII. The prevalence of catechin and epicatechin in chPP-L indicates that they form weak interactions with pectins. chPS-L1 and chPS-L2 enhanced ß2-integrin expression on neutrophils, inducing ROS generation and macrophage NO production. Both the effects on ß2-integrin and high complement fixation activity of chPS-L1 were proposed for its inhibitory action against PMA- and OZP-activated ROS formation. This, together with suppression of NO generation after co-stimulation with chPS-L1 and LPS, suggested anti-inflammatory activity of studied pectins. Lavender polysaccharides expressed intestinal Peyer’s patch immunomodulating activity.

doi:10.1038/s41598-017-02382-z

Abstract

Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBDCHIT1). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBDCHIT1 domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser.

doi:10.1016/j.tet.2017.05.057

Abstract

We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFA-mediated cyclization of a linear precursor containing three side chains. The introduction of a triphenylmethyl-protected thiol into carboxylic acid containing building blocks through sulfa Michael additions onto α,β-unsaturated hexafluoroisopropyl esters is described. The stereoselectivity of the bicycle formation was found to be somewhat lower than that previously reported for analogous 3,6,8-trisubstituted scaffolds. Moreover, the configuration of the linear precursor directs the stereochemical outcome of the cyclization differently when the R1 side chain is positioned on C2 in the bicycles (present work) instead of C3 (previous work). Tripeptidomimetic compounds based on the new scaffold were synthesized and evaluated for antagonistic potency toward CXCR4, and one compound (45a) displayed similar activity to earlier reported 3,6,8-tripeptidomimetic bicycles.

doi:10.1039/C6CP08506G

Abstract

Mixtures of surfactants and medium chained alcohols display an anomalous phase behaviour, with the formation of swollen micelles in mid-range surfactant concentrations, which transition into larger non-swollen aggregates when the surfactant concentration increases above a critical point. These alcohols also affect the adsorption behaviour of the surfactants. In this study, intermolecular proximities are measured for such systems by 1H–1H NMR dipolar correlation experiments, giving molecular localizations. The medium chained 1-heptanol and an anionic surfactant sodium dodecyl sulphate (SDS) are studied, both solubilized and adsorbed on alumina. Nuclear Overhauser Effect Spectroscopy (NOESY) shows that 1-heptanol localizes in both the palisade layer and in the core of SDS micelles when the 1-heptanol : SDS mole ratio increases beyond 2. The micelle diameter then increases with increasing 1-heptanol : SDS mole ratios due to more 1-heptanol partitioning in the micelle interior. When the micelle diameter increases beyond ∼6 nm, some SDS moves into the micelle interior, which may be a driving force for the structural transition at higher SDS concentrations. After being adsorbed on alumina, 1H–1H double-quantum magic angle spinning (DQ MAS) shows that SDS/1-heptanol bilayers are formed where 1-heptanol localizes in the palisade layer only, but with slightly different localizations compared to that in micelles. Three different 1-heptanol environments are identified on the surface by 2H NMR using 2H labelled 1-heptanol. However, in contrast to in solution, no 1-heptanol adsolubilizes in the bilayer interior.

doi:10.1016/j.carres.2017.02.003

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7–22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42–1298 mg per L of culture; 3.8–11.6 mg per OD600nm unit) as in shaking flasks, and showing the scale-up potential of the system.

doi:10.22203/ecm.v033a06

Abstract

Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1β. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1β. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1β, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells.

doi:10.1016/j.orggeochem.2016.08.016

Abstract

Four hexacyclic oleananes having characteristic mass spectra with an intense fragment at m/z 325 were found in Late Cretaceous/Tertiary terrigenous oils and immature source rocks containing 18α-oleanane and other saturated pentacyclic compounds of higher plant origin. They are sometimes as abundant as 18α-oleanane itself. They were not found in oils without oleanane. The major hexacyclic oleanane, comprising 50-80% of the hexacyclic oleananes, was isolated using high performance liquid chromatography (HPLC) and characterized using nuclear magnetic resonance spectroscopy (NMR) as 18α-oleanane with the Me group at C-10 shifted to C-9 and with the Me at C-14 creating a new methylene bridge to C-10. Hexacyclic oleananes were not visible in m/z 191 chromatograms. They are most effectively detected by way of GC-MS-MS using the m/z 410 → 325 transition. Since few other compounds produce any m/z 410 → 325 signal, they may sometimes be easier to detect than regular and rearranged oleananes in samples with a low concentration of angiosperm markers. Natural precursor products have not been identified among plant triterpenoids, but might be related to (poly)functionalized oleanoids. Oleanoids hydroxylated at C-27 are obvious precursors. Oxic/clay-rich depositional environments seem to favour the formation of hexacyclic oleananes. A series of 2-alkylated oleananes had previously been characterized using NMR. Their hexacyclic counterparts were tentatively assigned in this study.

doi:10.1021/acs.jafc.6b02853

Abstract

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

doi:10.1021/acs.energyfuels.6b00799

Abstract

A series of novel C33–C35 hexacyclic benzohopanes (C33b-C35b) were identified in 39 samples of coal extracts and 39 crude oils of different ages from all over the world. C33b and C34b homologues were isolated, and their structures were determined by nuclear magnetic resonance. The structure of C35b benzohopane was proposed based on the mass spectrum and its similarity with the mass spectra of structurally defined C33b and C34b homologues. The structures of the C33b-C35b hexacyclic benzohopanes are closely related to isohopanes; both groups are typical for terrestrial organic matter and can be useful in the correlation analysis. A possible pathway of formation of these novel benzohopanes and their hopanoid precursors with an additional branch in the aliphatic side chain is proposed. C33b–C35b hexacyclic benzohopanes are stable up to the maturity level corresponding to random vitrinite reflectance (Rr) of ∼0.80%, which was demonstrated by analyzing the samples of different maturity and by the maturation simulation experiments: hydrous pyrolysis of two bituminous coals (Rr = 0.55 and 0.59%) and pyrolysis of an extracted bituminous coal (Rr = 0.56%) and its asphaltenes. This represents a confirmation that the formation of these novel benzohopanes is related to specific depositional conditions and microbial activity during diagenesis. Mature samples (Rr ≥ 0.8%) and hydrous pyrolysate of the bituminous coals (Rr < 0.60%) obtained at 330 °C show a distinct distribution of benzohopanes in comparison to immature and moderately mature samples, which is characterized by a low abundance of the “b” series benzohopanes and the presence of regular and numerous other benzohopane isomers. The latter most likely represent isomers of regular and novel benzohopanes with different substitution patterns on the aromatic ring. This isomerization of alkyl groups attached to the aromatic ring, leading to the formation of thermodynamically more stable isomers, is a well-known maturation scenario so far reported in the series of alkylated naphthalenes, phenanthrenes, and dibenzothiophenes. Therefore, in the same way, a distribution of benzohopanes can indicate thermal maturity. In addition to the novel benzohopanes, three series (2α, 2β, and 3β) of their methylated derivatives were identified in numerous samples. Finally, a novel C35 heptacyclic benzohopane with an additional cyclopentane ring was also observed in the studied samples, and its structure was tentatively identified based on the mass spectrum. Opposite to the hexacyclic C33b–C35b benzohopanes, the formation of the C35 heptacyclic benzohopane does not require a specific hopanoid precursor with two branches in the side chain. Therefore, this compound seems to have less geochemical significance than the new hexacyclic benzohopanes.

doi:10.1073/pnas.1602566113

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

doi:10.1007/s12104-016-9683-x

Abstract

The apo-form of the 23.3 kDa catalytic domain of the AA9 family lytic polysaccharide monooxygenase NcLPMO9C from Neurospora crassa has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments of this LPMO.

doi:10.1016/j.carbpol.2016.03.052

Abstract

A comprehensive characterization of polysaccharides from the chanterelle was performed. Experiments included both linkage analysis by methylation and GC-MS, monosaccharide composition analysis by methanolysis, SEC-MALLS and several NMR experiments including COSY, HSQC, TOCSY, HSQC-TOCSY, NOESY, and HMBC. A 671kDa (1→6)-linked α-d-mannan with single and short (1→2)-linked side chains (WCcF1b) was isolated from the hot water extract, after protease treatment and fractionation by size exclusion chromatography. The hot 1M NaOH extract contained two types of β-glucan; a water soluble (1→6)-linked β-d-glucan with single and short (1→3)-linked β-glucan side chains (ACcSw) and a less branched (1→3)-linked β-d-glucan (ACcIw/ACcId), proposed to contain short side chains in O-4. Chemical shifts assignments of the α-mannan and β-glucan are presented as well as chemical shift values obtained from a (1→6)/(1→3)-β-d-glucan derived from Saccharomyces cerevisiae which was used as reference.

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