Preventive and curative glycoside kaempferol treatments attenuate the TH2-driven allergic airway disease

Asthma is a chronic respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). One strategy to treat allergic diseases is the development of new drugs. Flavonoids are compounds derived from plants and are known to have antiallergic, anti-inflammatory, and antioxidant properties. To investigate whether the flavonoid kaempferol glycoside 3-O-[beta-D-glycopiranosil-(1 -> 6)-alpha-L-ramnopiranosil]-7-O-alpha-L-ramnopiranosil-kaempferol (GRRK) would be capable of modulating allergic airway disease (AAD) either as a preventive (GRRK P) or curative (GRRK C) treatment in an experimental model of asthma. At weekly intervals, BALB/c mice were subcutaneously (sc) sensitized twice with ovalbumin (OVA)/alum and challenged twice with OVA administered intranasally. To evaluate any preventive effects GRRK was administered 1 h (hour) before each OVA-sensitization and challenge, while to analyze the curative effects mice were first sensitized with OVA, followed by GRRK given at day 18 through 21. The onset: of AAD was evaluated 24 h after the last OVA challenge. Both treatments resulted in a dose-dependent reduction in total leukocyte and eosinophil counts in the bronchoalveolar lavage fluid (BAL). GRRK also decreased CD4(+), B220(+), MHC class II and CD40 molecule expressions in BAL cells. Histology and lung mechanic showed that GRRK suppressed mucus production and ameliorated the AHR induced by OVA challenge. Furthermore, GRRK impaired Th2 cytokine production (IL-5 and IL-13) and did not induce a Th1 pattern of inflammation. These findings demonstrate that GRRK treatment before or after established allergic lung disease down-regulates key asthmatic features. Therefore. GRRK has a potential clinical use for the treatment of allergic asthma. (C) 2009 Elsevier B.V. All rights reserved.

Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2`,3`-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2`,3`-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry

Catalytic mechanism of inulinase from Arthrobacter sp S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K-m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K-m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Crystalline structure of mangiferin, a C-glycosyl-substituted 9H-xanthen-9-one isolated from the stem bark of Mangifera indica

The crystalline structure of mangiferin (= 2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one; 1), a biologically active xanthenone C-glycoside, isolated from the stem bark of Mangifera indica (Anacardiaceae), was unambiguously determined by single-crystal X-ray diffraction (XRD). The crystal structure is summarized as follows: triclinic, P1, a = 7.6575(5), b = 11.2094(8), c = 11.8749(8) angstrom, alpha = 79.967(5), beta = 87.988(4), gamma = 72.164(4)degrees, V = 955.3(1) angstrom(3), and Z = 2. The structure also shows two molecules in the asymmetric unit cell and five crystallization H2O molecules. The packing is stabilized by several intermolecular H-bonds involving either the two symmetry-independent mangiferin molecules 1a and 1b, or the H2O ones.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES double dagger. Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.

Role of the triad N46, S106 and T107 and the surface charges in the determination of the acidic pH optimum of digestive lysozymes from Musca domestica

Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106-T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of 050 (catalytic nucleophile), whereas those residues are replaced by V109-A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D-S106V-T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1 unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces. (C) 2010 Elsevier Inc. All rights reserved.

Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an encloglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections. (C) 2009 Elsevier Ltd. All rights reserved.

Thermal behavior and structural properites of plant-derived eugenyl acetate

Eugenol is an allyl chain-substituted guaiacol in the biosynthesized phenylpropanoid compound class derived from Syzygium aromaticum L. and widely used in folk medicine. Nonetheless, its pharmacological use is limited by some problems, such as instability when exposed to light and high temperature. In order to enhance stability, the eugenol molecule was structurally modified, resulting in eugenyl acetate. The eugenyl acetate`s thermal behavior and crystal structure was then characterized by differential scanning calorimetry (DSC) and X-ray diffraction (XRD) and compared to a commercial sample.