Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer

Conclusions

What is the function of parent–child argumentation? This chapter intends to answer the main research question that has guided the study presented in this volume and open a discussion for future research on this topic. In the first part, the chapter provides a detailed overview of the main findings of the analysis of parent–child argumentative discussions during mealtime. The role played by parents and children in the inception and development of argumentation, and the types of conclusions of their argumentative discussions are described. Subsequently, two educational targets achieved by parents and children through their argumentative interactions are presented and critically discussed. In the last part, new open questions that should guide future investigation to expand our knowledge of the role and function of argumentation between parents and children are proposed

Structural characterization and interaction studies of humanlipocalin-type prostaglandin D synthase (L-PGDS)

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precursor of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central and peripheral nervous system and it is involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as \u3b2-trace protein, the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, testis and prostate, endothelial cells, placenta and heart tissue and even in macrophages infiltrated in atherosclerotic plaques. In these tissues it participates in many physiological activities as well as in the response to diseases. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. In this work we use recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction and test its affinity for several ligands using Surface Plasmon Resonance (SPR). Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion and hydrophobic interaction chromatography. Large and highly ordered crystals were used to collect X-ray diffraction data using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement method was used to solve the phase problem. In the electron density maps an unidentified density was observed apparently interacting with lysine 59 inside the L-PGDS-C65A cavity; the foreign molecule is probably PEG, an additive present in the crystallization liquors. This hypothesis is supported by the fact that the L-PGDS-C65A/K59A crystals, which grow without PEG, show a completely free protein cavity. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. Another crystal form was obtained by mixing L-PGDS-C65A/K59A with the amyloid \u3b2 peptide (1-40). Although the amyloid \u3b2 peptide is not visible in the maps, the packing of the protein molecules has changed in the presence of the peptide suggesting interaction of the two molecules. Wild type L-PGDS small crystals were recently obtained and will be tested as soon beam time at a synchrotron source becomes available. SPR experiments are also in progress and will be used to verify interaction of L-PGDS with PEG, the amyloid \u3b2 peptide and other ligands and to determine their binding constants

4-amino-1-(2-deoxy-beta-D-ribofuranosyl)-6,7-dihydro-1H,5H-cyclopentapyrimidine-2-one

The crystal structure of C12H17N304 has been determined. This modified base is in a syn conformation with respect to the deoxyribose sugar, which adopts a distorted C3'/04'-endo pucker

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS)

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS)

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS

The alpha-pyridyl nucleoside analogue 2-bromo-5-(2-deoxy-alpha-D-ribofuranosyl)pyridine

The structure of the title compound, C10H12BrNO3, is reported. The ribose sugar has a C3'-endo pucker and the exocyclic torsion angle 05'--C5'---C4'--C3' adopts a gauche + value of 61.0 (9) °

Free-breathing Pulmonary MR Imaging to Quantify Regional Ventilation

Purpose: To measure regional specific ventilation with free-breathing hydrogen 1 (1H) magnetic resonance (MR) imaging without exogenous contrast material and to investigate correlations with hyperpolarized helium 3 (3He) MR imaging and pulmonary function test measurements in healthy volunteers and patients with asthma. Materials and Methods: Subjects underwent free-breathing 1H and static breath-hold hyperpolarized 3He MR imaging as well as spirometry and plethysmography; participants were consecutively recruited between January and June 2017. Free-breathing 1H MR imaging was performed with an optimized balanced steady-state free-precession sequence; images were retrospectively grouped into tidal inspiration or tidal expiration volumes with exponentially weighted phase interpolation. MR imaging volumes were coregistered by using optical flow deformable registration to generate 1H MR imaging-derived specific ventilation maps. Hyperpolarized 3He MR imaging- and 1H MR imaging-derived specific ventilation maps were coregistered to quantify regional specific ventilation within hyperpolarized 3He MR imaging ventilation masks. Differences between groups were determined with the Mann-Whitney test and relationships were determined with Spearman (ρ) correlation coefficients. Statistical analyses were performed with software. Results: Thirty subjects (median age: 50 years; interquartile range [IQR]: 30 years), including 23 with asthma and seven healthy volunteers, were evaluated. Both 1H MR imaging-derived specific ventilation and hyperpolarized 3He MR imaging-derived ventilation percentage were significantly greater in healthy volunteers than in patients with asthma (specific ventilation: 0.14 [IQR: 0.05] vs 0.08 [IQR: 0.06], respectively, P \u3c .0001; ventilation percentage: 99% [IQR: 1%] vs 94% [IQR: 5%], P \u3c .0001). For all subjects, 1H MR imaging-derived specific ventilation correlated with plethysmography-derived specific ventilation (ρ = 0.54, P = .002) and hyperpolarized 3He MR imaging-derived ventilation percentage (ρ = 0.67, P \u3c .0001) as well as with forced expiratory volume in 1 second (FEV1) (ρ = 0.65, P = .0001), ratio of FEV1 to forced vital capacity (ρ = 0.75, P \u3c .0001), ratio of residual volume to total lung capacity (ρ = -0.68, P \u3c .0001), and airway resistance (ρ = -0.51, P = .004). 1H MR imaging-derived specific ventilation was significantly greater in the gravitational-dependent versus nondependent lung in healthy subjects (P = .02) but not in patients with asthma (P = .1). In patients with asthma, coregistered 1H MR imaging specific ventilation and hyperpolarized 3He MR imaging maps showed that specific ventilation was diminished in corresponding 3He MR imaging ventilation defects (0.05 ± 0.04) compared with well-ventilated regions (0.09 ± 0.05) (P \u3c .0001). Conclusion: 1H MR imaging-derived specific ventilation correlated with plethysmography-derived specific ventilation and ventilation defects seen by using hyperpolarized 3He MR imaging. © RSNA, 2018 Online supplemental material is available for this article

Structural and biophysical studies on the lectin domain of GalNAc-T6 for therapeutic applications

The expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Gal\u3b21\u20133GalNAc; T-antigen) and the Lewis antigen (sialyl-T-antigen) is a characteristic feature observed on almost all malignant epithelial cells. Therefore, there is a particular interest in their application not only as prognostic markers but also as therapeutic targets [1]. These antigens can be recognized by lectins, a group of highly specific carbohydrate-binding proteins that have been proposed as useful tools for antitumor drug-targeting [2].The three-dimensional structure of several lectins with antitumor properties has been determined in our laboratory by X-ray crystallography. N-\u3b1-acetylgalactosaminyltransferase-6 (GalNAc-T6) is an enzyme present also in humans which contains a catalytic domain and a lectin domain with a binding site for N-acetylgalactosamine (GalNAc), one of the saccharides exposed by cancer cells (Tn-antigen). Unlike other lectins with these properties, the lectin domain of GalNAc-T6 presents a structural fold found also in other human proteins, unlocking the opportunity to use protein engineering tools to design new anticancer therapeutics [3]. The three-dimensional structure of GalNAc-T6 has not been determined so far, neither has been its substrate specificity. Therefore, the production of a recombinant form containing only the lectin domain can contribute to these two critical points that need to be considered to evaluate its possible use in cancer therapies. The lectin domain of this enzyme was expressed by cloning the C-terminal portion of the DNA coding sequence and introducing it into Pichia pastoris for its recombinant production. Biophysical methods such as spectrofluorimetry and isothermal titration calorimetry were used to analyze the ability of the engineered protein to bind the T-antigen monosaccharides. The binding dissociation constant (Kd) of the protein-carbohydrate interaction was determined. The stability of the protein was also studied through its thermodynamic parameters of unfolding using differential scanning calorimetry. Crystallization screenings were set up using a broad variety of precipitants in order to produce crystals to be used to study the three-dimensional structure of the engineered protein using X-ray diffraction. The crystals that were grown were taken to the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to carry out the diffraction experiments. Although we were able to collect data up to a resolution of 2.8 \uc5 (854,648 reflections) all the crystals we have examined so far were found to be twinned making the assignment of a definitive space group uncertain. We are currently working on correcting this problem using both the appropriate software and attempting to grow better crystals. Our goal is to produce an engineered human protein that specifically recognizes cancer specific carbohydrates and is thus suitable for protein therapeutics applied in drug-delivery methods for cancer treatment. The present structural and biophysical data are the prerequisite for future studies regarding the biological and clinical properties of the lectin. [1] Stowell, S. R. Tongzhong J. and Cummings R. D. Protein Glycosylation in Cancer. Annu Rev Pathol 2015. 10: 473\u2013510. [2] Sharon, N., and Lis, H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004. 14: 53\u201362. [3] Berois, N., Mazal, D. et al. UDP-N-Acetyl-D-Galactosamine: N-acetylgalactosaminyltransferase-6 as a New Immunohistochemical Breast Cancer Marker. Journal of Histochemistry & Cytochemistry. 2006. 54(3): 317\u2013328