Guilherme L. Sassaki

An α-Glucan of Pseudallescheria boydii Is Involved in Fungal Phagocytosis and Toll-like Receptor Activation

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an α-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that α-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked α-d-Glcp residues substituted at position 6 with α-d-Glcp branches. Soluble α-glucan, but not β-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when α-glucan from conidial surface was removed by enzymatic treatment with α-amyloglucosidase, thus indicating an essential role of α-glucan in P. boydii internalization by macrophages. α-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with α-amyloglucosidase. Finally, α-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and α-glucan could be a target for intervention during fungal infections.

Application of acetate derivatives for gas chromatography–mass spectrometry: Novel approaches on carbohydrates, lipids and amino acids analysis

The structure of glycoconjugates has been determined by several chromatographic methods, however gas chromatography-mass spectrometry (GC-MS) has been widely used to identify and quantify the volatile trimethylsilyl and fluoroacyl derivatives. Adapting the reduction/acetylation strategies, we had performed the derivatization of all monosaccharide class, as well as amino acids and OH-fatty acids as from different glycoconjugates. Uronic acids gave characteristic ions at m/z 143, 156 and 173, and 19 amino acids derivatives, gave molecular ions [M]+ and daughter ions of [M-59]+ and [M-43]+ on electron impact (EI)-MS, which provide their rapid identification.

An α-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and TLR activation

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an α-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that α-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked α-d-Glcp residues substituted at position 6 with α-d-Glcp branches. Soluble α-glucan, but not β-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when α-glucan from conidial surface was removed by enzymatic treatment with α-amyloglucosidase, thus indicating an essential role of α-glucan in P. boydii internalization by macrophages. α-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with α-amyloglucosidase. Finally, α-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and α-glucan could be a target for intervention during fungal infections.

HPLC/ESI-MS and NMR analysis of flavonoids and tannins in bioactive extract from leaves of Maytenus ilicifolia

Maytenus ilicifolia is an important plant in Brazilian folk medicine, used in many gastric disorder treatments. Low molecular weight components present in the leaves have been characterized as afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin and epigallocatechin, as detected by HPLC and ESI-MS. Condensed tannins have also been found, consisting of di-, tri-, tetra-, and penta-, hexa, and heptamers. ESI-MS analyses were performed in positive and negative ionization modes, and in contrast with other investigations, negative ionization improved sensitivity for obtaining molecular ions. Moreover, the tandem-MS profile with negative detection provided characteristic fragments, such as those found at m/z 543 [(epi)afzelechin-(epi)afzelechin], m/z 561 [(epi)afzelechin-(epi)catechin], and m/z 577 [(epi)catechin-(epi)catechin] or [(epi)afzelechin-(epi)gallocatechin]. The analysis of the fragments also indicated the presence of additional ether linkage between C2 and C7, present in A-Type proanthocyanidins, and were identified at m/z 559 [(epi)afzelechin-(epi)catechin], m/z 575 [(epi)catechin-(epi)catechin] or [(epi)afzelechin-(epi)gallocatechin] and at m/z 591, [(epi)catechin-(epi)gallocatechin]. CID-fragmentation was used for tannins sequencing, as well as 3D NMR HMQC-TOCSY and COSY, which provided fingerprint assignments for identification of cathechin at delta 4.55/82.1 (H-2/C-2), 3.96/68.1 (H-3/C-3) and 2.82-2.50/27.7 (H-4a-H-4b/C-4), and epicathechin delta 4.78/79.1 (H-2/C-2), 4.15/66.7 (H-3/C-3) and 2.82-2.73/28.5 (H-4a-H-4b/C-4). Since HMQC-TOCSY gives a high resolution heteronuclear connectivity, it is useful for identification of other cis-trans isomers present in complex flavonoid mixtures.

Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid-liquid partition and two-dimensional liquid chromatography (size exclusion × reversed phase).

Green and black teas (Camellia sinensis) contain compounds ranging from simple phenolics to complex glycosides, many of which have well-recognized health benefits. Here, we describe two methodologies aiming to achieve a comprehensive analysis of hydro-alcoholic extracts of C. sinensis. In the first step, the extracts were partitioned in water, n-butanol, ethyl acetate and chloroform to separate the compounds according to their polarity, yielding less complex samples to be analyzed by ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Additionally, a comprehensive two dimensional liquid chromatography (2D-LC) technique, employing size exclusion chromatography (SEC) × reversed phase (BEH-C18) was developed. The following compounds were identified on the basis of retention time, UV-spectra and MS fragmentation patterns: catechins, theaflavins and their gallate derivatives; kaempferol, quercetin and myricetin mono-, di-, tri- and tetraglycosides; esters of quinic acid and gallic or hydroxycinnamic acids; purine alkaloids, such as caffeine and theobromine and many lipids. Additionally, there were many novel compounds that were previously undescribed, such as saponin isomers and gallic acid esters of four glycosides of myricetin, quercetin and kaempferol.

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.

Structure and degree of polymerisation of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni

Fructooligosaccharides have been isolated from roots and leaves of Stevia rebaudiana, by hot aqueous extraction, followed by precipitation with ethanol. Their structure has been determined using methylation and NMR analysis, MALDI-TOF, and ESI-MS. Fructooligosaccharides contained almost exclusively (2→1)-linked β-fructofuranosyl, with terminal α-glucopyranosyl and β-fructofuranosyl units. MALDI-TOF and ESI-MS analyses showed the wide range of degree of polymerisation (DP) present in various extracts. From roots and leaves, three different fractions gave profiles of homologous series, with DPs ranging up to 17 with MALDI-TOF and 19 using ESI-MS. These inulin-type fructooligosaccharides were the major component of extracts from S. rebaudiana roots and significant components from the leaves.

Heart-cutting two-dimensional (size exclusion × reversed phase) liquid chromatography–mass spectrometry analysis of flavonol glycosides from leaves of Maytenus ilicifolia

Two-dimensional liquid chromatography-electrospray ionization mass spectrometry was employed to analyze flavonol glycosides present in leaves of Maytenus ilicifolia, frequently used in traditional Brazilian medicine. Since they contain many flavonol glycosides, including isomers, one-dimensional liquid chromatography did not give complete separation and identification, yielding overlapping of compounds with different molecular weights. Thus, employing size exclusion chromatography in the first and reversed phase in the second dimension, a great number of flavonol glycosides could be identified and its relative abundances determined. The majority of glycosides contained kaempferol or quercetin as aglycones, and glycosides with previously unreported structures were also present and characterized.

Methylation-GC-MS analysis of arabinofuranose- and galactofuranose-containing structures: rapid synthesis of partially O-methylated alditol acetate standards

Arabinose and galactose were treated with MeOH containing traces of H2SO4 or HCl at 25 ◦ C to give mixtures of their methyl alpha- and beta-furanosides, as shown by 1D and 2D nuclear magnetic resonance (NMR). Oxidation of the Me alpha,beta-Araf mixture with NaIO4 preferentially oxidised the beta-isomer, to give pure Me alpha-Araf . Each product was progressively O-methylated using the Purdie reagent (MeI/Ag2O) at 25 ◦ C and resulting mixtures of partially methylated glycosides (PMGs) were rapidly assayed by thin layer chromatography (TLC) first to favour higher yields of mono-O-methyl derivatives and later for products with higher degrees of methylation. The products were converted to complex mixtures of partially O-methylated alditol acetate derivatives (PMAAs) by successive hydrolysis, reduction with NaBD4, and acetylation. These can be used as gas chromatography-mass spectrometry (GC-MS) standards in methylation analysis of complex carbohydrates containing arabinofuranosyl and galactofuranosyl units. Of particular interest were the retention times and electron impact MS of the difficult to prepare alditol acetates of 5,6-Me2Gal, 2,5-Me2Gal, 2,5,6-Me3Gal, 3,5,6-Me3Gal, 5-MeAra, 2,5-Me2Ara, and 3,5-Me2Ara. The relative reactivities of hydroxyl groups for mixtures of Me alpha- and Me beta-Galf were HO-2 > HO-3 > HO-6 > HO-5, that of Me alphaand Me beta-Araf HO-2 > HO-3 > HO-5, and that of Me alpha-Araf HO-2 > HO-3 ≥ HO-5.

Lactarius rufus (1→3),(1→6)-β-d-glucans: Structure, antinociceptive and anti-inflammatory effects

Medicinal health benefits uses of edible as well as non-edible mushrooms have been long recognized. The pharmacological potential of mushrooms, especially antitumor, immunostimulatory and anti-inflammatory activities has been documented. Wild ectomycorrhizal mushroom, Lactarius rufus had the anti-inflammatory and antinociceptive potential of their polysaccharides evaluated using the formalin model. Two structurally different (1→3),(1→6)-linked β-D-glucans were isolated from fruiting bodies. Soluble (FSHW) β-D-glucan 1-30 mg kg(-1) produced potent inhibition of inflammatory pain caused by formalin when compared with the insoluble one (IHW), suggesting that solubility and/or branching degree could alter the activity of β-glucans. Their structures were determined using mono- and bi-dimensional NMR spectroscopy, methylation analysis, and controlled Smith degradation. They were β-D-glucans, with a main chain of (1→3)-linked Glcp residues, substituted at O-6 by single-unit Glcp side chains (IHW), on average to every fourth residue of the backbone, or by mono- and few oligosaccharide side chains for soluble β-glucan.