Fluorescent labelled thiourea-bridged glycodendrons

GlcNAc-coated glycodendrimers, which are polyvalent glycomimetics, display strong in vitro affinity for the rat natural killer cell protein-1A (NKR-P1A), a C-type lectin-like receptor of natural killer (NK) cells in rats, humans and some strains of mice. Administration of these compounds in vivo results in a substantial increase in the antitumour activity with involvement of the natural cell immunity. To clarify the in vitro and in vivo fate of these molecules, we synthesized labelled glycodendron analogues of the previously studied glycodendrimers. Labelling with fluorescent tags enabled the localization of the glycodendrons in white blood cells, tumours and other tissues by using different imaging techniques such as fluorescence and confocal microscopy. These studies are useful for probing the mechanism of action and fate of artificial ligands and the cell receptors involved.     

Re-Evaluation of Binding Properties of Recombinant Lymphocyte Receptors NKR-P1A and CD69 to Chemically Synthesized Glycans and Peptides

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-d-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.     

What function for human lithostathine?: structural investigations by three-dimensional structure modeling and high-resolution NMR spectroscopy

Human lithostathine is a 144-residue protein, expressed in variousorgans and pathologies. Several biological functions have beenproposed for this protein. Among others, inhibition of nucleationand growth of CaCO₃ crystals in the pancreas and bacterial aggregationhas retained attention, because lithostathine presents highsequence similarities with calcium-dependent (or C-type) lectins.To study its structure-function relationship and compare itwith that of C-type lectins, we have built a model for lithostathine.This model is derived from the only two C-type lectins of knownstructures: rat mannose binding protein and human E-selectin.An original strategy, inspired by that proposed by Havel andSnow, was designed for model building. We have undertaken NMRstudies on the natural protein. Although complete structuredetermination has not yet been achieved, the NMR studies didconfirm the main characteristics of the model. From analysisof the proposed model, we concluded that lithostathine is notexpected to present sugar- or calcium-binding properties. Therefore,the mechanisms of bacterial aggregation and inhibition of CaCO₃nucleation and growth have not yet been elucidated.     

Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms

One of the challenges of science in disease prevention is optimizing drug and vaccine delivery. Until now, many strategies have been employed in this sector, but most are quite complex and labile. To overcome these limitations, great efforts are directed to coupling drugs to carriers, either of natural or synthetic origin. Among the most studied cell carriers are antigen-presenting cells (APCs), however, red blood cells (RBCs) are positioned as attractive carriers in drug delivery due to their abundance and availability in the body. Furthermore, fish RBCs have a nucleus and have been shown to have a strong involvement in modulating the immune response. In this study, we evaluated the binding of three peptides to rainbow trout RBCs, two lectin-like peptides and another derived from Plasmodium falciparum membrane protein, in order to take advantage of this peptide-RBCs binding to generate tools to improve the specificity, efficacy, immunostimulatory effect, and safety of the antiviral therapeutic or prophylactic administration systems currently used.     

Systemic Lectin-Glycan Interaction of Pathogenic Enteric Bacteria in the Gastrointestinal Tract

Microorganisms, such as bacteria, viruses, and fungi, and host cells, such as plants and animals, have carbohydrate chains and lectins that reciprocally recognize one another. In hosts, the defense system is activated upon non-self-pattern recognition of microbial pathogen-associated molecular patterns. These are present in Gram-negative and Gram-positive bacteria and fungi. Glycan-based PAMPs are bound to a class of lectins that are widely distributed among eukaryotes. The first step of bacterial infection in humans is the adhesion of the pathogen’s lectin-like proteins to the outer membrane surfaces of host cells, which are composed of glycans. Microbes and hosts binding to each other specifically is of critical importance. The adhesion factors used between pathogens and hosts remain unknown; therefore, research is needed to identify these factors to prevent intestinal infection or treat it in its early stages. This review aims to present a vision for the prevention and treatment of infectious diseases by identifying the role of the host glycans in the immune response against pathogenic intestinal bacteria through studies on the lectin-glycan interaction.     

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.     

Fold recognition and molecular modeling of a lectin-like domain in UDP- GalNac:polypeptide N-acetylgalactosaminyltransferases

By use of threading methods, the C-terminal region of uridine diphospho- N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-transferases) was predicted to have the same fold as the lectin-domain of the plant cytotoxins ricin and abrin-a, for which crystal structure are available. The sequence identities are very low. Nevertheless, the amino acids involved in the hydrophobic core essential for the structure stability and the cysteine residues are conserved. In addition, the amino-acids involved in carbohydrate binding are conserved in ppGalNAc-transferases. The extra C-terminal domain of these enzymes is therefore a putative glycan-binding domain. A model of the lectin-like domain of human ppGalNAc-transferase T1 was built using knowledge based methods. Geometry optimization of the complex with galactose allowed prediction that this domain could bind this monosaccharide. However, the interaction seems to be rather weak, and at the moment there is no evidence that ppGalNAc-transferases displays a lectin activity in vivo.      

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.     

Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens such as Mycobacterium tuberculosis. As an expansion of our previous work, a library of 6,6′-amide and sulfonamide α,α-d -trehalose compounds with various substituents on the aromatic ring was synthesized efficiently in good to excellent yields. These compounds were evaluated for their ability to activate the human C-type lectin receptor Mincle by the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure–activity relationship (SAR) of these novel trehalose diamides and sulfonamides revealed that aryl amide-linked trehalose compounds demonstrated improved activity and relatively high potency cytokine production compared to the Mincle ligand trehalose dibehenate adjuvant (TDB) and the natural ligand trehalose dimycolate (TDM) inducing dose-dependent and human-Mincle-specific stimulation in a HEK reporter cell line.     

Comparative analysis of putative agonist-binding modes in the human A1 adenosine receptor

A recent study reported a model of the human A(1) adenosine receptor and its agonist binding site, proposing two putative binding modes in the same binding site for the natural agonist, adenosine. The present work investigates the flexibility of this binding site by exhaustive exploration with the natural agonist and with three other adenosine derivatives: N6-cyclopentyladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and 5'-N-ethylcarboxamidoadenosine (NECA). Our aim was to find a common binding mode for agonists that would explain the role in the binding process of the different substitutions allowed at the 2, N6, and 5' positions of adenosine. This problem was addressed through docking simulations, molecular dynamics studies, and estimations of the ligand-binding free energy with both the AUTODOCK scoring function and the linear interaction energy (LIE) approach. The results point to a single receptor-binding position that explains the effects of the different chemical modifications on the adenosine derivatives considered here.     

Influence of zirconia raw materials on the development of DeNOx monolithic catalysts

Pd-zirconia-based monolithic catalysts were prepared with various commercial zirconia raw materials and a natural magnesium silicate binder, sepiolite, for the selective catalytic reduction (SCR) of NO with CH₄ in oxygen excess. The different textural properties, metastable tetragonal zirconia phase stability, surface acidity, Pd dispersion and catalytic properties of these monoliths were compared to select the most suitable structured catalyst for NO_x control in natural gas-fired power plants. The influence of operating temperature in the two reactions, NO reduction and CH₄ combustion, with the monolithic catalysts was determined. A 0.4 wt.% Pd-zirconia catalyst, manufactured from a sulphated zirconium hydroxide raw material, was selected as the most appropriate in the reaction under study, reaching a maximum NO conversion at 400 °C.     

Modeling the Combining Site of the Human Asialoglycoprotein Receptor

A model for the carbohydrate recognition domain (CRD) and combining site of the human asialoglycoprotein (ASGP) receptor has been computed on the basis of the close sequence homology with the mannose-binding lectin (MBP), whose three-dimensional structure in complex with a ligand has been determined by crystallographic methods (Weis, W.I.; Drickamer, K.; Hendrickson, W.A. Nature 1992, 360: 127). Within the limitations of modeling methods, the model is compatible with data on ligand binding of the family C-type lectins, of which the MBP and the ASGP receptor are members. The model derived can serve as a guide for designing site-directed mutagenesis experiments in order to further elucidate the origins of specificity of the ASGP receptor toward galactose. In particular, the model focuses attention on the possible role of position 207 (MBP sequence numbering) in promoting galactose binding.     

Myeloperoxidase-Oxidized LDL Activates Human Aortic Endothelial Cells through the LOX-1 Scavenger Receptor

Cardiovascular disease as a result of atherosclerosis is a leading cause of death worldwide. Atherosclerosis is primarily caused by the dysfunction of vascular endothelial cells and the subendothelial accumulation of oxidized forms of low-density lipoprotein (LDL). Early observations have linked oxidized LDL effects in atherogenesis to the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scavenger receptor. It was shown that LOX-1 is upregulated by many inflammatory mediators and proatherogenic stimuli including cytokines, reactive oxygen species (ROS), hemodynamic blood flow, high blood sugar levels and, most importantly, modified forms of LDL. Oxidized LDL signaling pathways in atherosclerosis were first explored using LDL that is oxidized by copper (Cuox-LDL). In our study, we used a more physiologically relevant model of LDL oxidation and showed, for the first time, that myeloperoxidase oxidized LDL (Mox-LDL) may affect human aortic endothelial cell (HAEC) function through the LOX-1 scavenger receptor. We report that Mox-LDL increases the expression of its own LOX-1 receptor in HAECs, enhancing inflammation and simultaneously decreasing tubulogenesis in the cells. We hypothesize that Mox-LDL drives endothelial dysfunction (ED) through LOX-1 which provides an initial hint to the pathways that are initiated by Mox-LDL during ED and the progression of atherosclerosis.     

Identification of tetranectin as a potential biomarker for metastatic oral cancer

Lymph node involvement is the most important predictor of survival rates in patients with oral squamous cell carcinoma (OSCC). A biomarker that can indicate lymph node metastasis would be valuable to classify patients with OSCC for optimal treatment. In this study, we have performed a serum proteomic analysis of OSCC using 2-D gel electrophoresis and liquid chromatography/tandem mass spectrometry. One of the down-regulated proteins in OSCC was identified as tetranectin, which is a protein encoded by the CLEC3B gene (C-type lectin domain family 3, member B). We further tested the protein level in serum and saliva from patients with lymph-node metastatic and primary OSCC. Tetranectin was found significantly under-expressed in both serum and saliva of metastatic OSCC compared to primary OSCC. Our results suggest that serum or saliva tetranectin may serve as a potential biomarker for metastatic OSCC. Other candidate serum biomarkers for OSCC included superoxide dismutase, ficolin 2, CD-5 antigen-like protein, RalA binding protein 1, plasma retinol-binding protein and transthyretin. Their clinical utility for OSCC detection remains to be further tested in cancer patients.     

[Gly14]-Humanin Prevents Lipid Deposition and Endothelial Cell Apoptosis in a Lectin-like Oxidized Low-density Lipoprotein Receptor-1-Dependent Manner

Oxidized low-density lipoprotein (Ox-LDL) may induce apoptosis and dysfunction of vascular endothelial cells, contributing to the initiation and development of atherosclerosis and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a central role in Ox-LDL uptake in the course of atherogenesis. Humanin (HN), a mitochondrial-derived peptide, was recently demonstrated to exert a protective role against endothelial dysfunction and Ox-LDL-induced progression of atherosclerosis. The HN analog HNGF6A (HNG) modulates cholesterol metabolism in macrophage RAW 264.7 cells. However, whether HNG affects Ox-LDL metabolism in endothelial cells is unknown. In this study, we investigated the effect of HNG on Ox-LDL accumulation in human umbilical vein endothelial cell (HUVEC) and its underlying mechanisms. HUVEC were preincubated with HNG for 1 h before addition of Ox-LDL. Total cholesterol content was measured by using a tissue total cholesterol assay kit and flow cytometry. Cell viability was measured by CCK8 assay. Protein content was examined by Western blot assays. Flow cytometry was used to identify apoptotic cells. Flow cytometry and tissue total cholesterol assays showed that HNG reduced Ox-LDL accumulation in HUVEC. In addition, HNG inhibited Ox-LDL-induced apoptosis of HUVEC. Western blot results showed that HNG reduced LOX-1 protein content. However, when LOX-1 was knocked down or inhibited, the effect of HNG in reducing Ox-LDL aggregation and apoptosis in HUVEC disappeared. Our study demonstrated that HNG reduces lipid aggregation and apoptosis in HUVEC in a LOX-1-dependent manner.     

Spectral properties of the second-order neurons in the dace retina

Spectral properties of the horizontal cells and the bipolar cells in the dace retina were studied under mesopic and/or photopic conditions. Four types of horizontal cells were identified: two L types (L1 and L2) and two C types (RB and RGB). The L1 cell has its spectral peak at about 590 nm, and the L2 cell peaks at about 630 nm. The wavelength at which a maximum hyperpolarization of the RB cell is produced is around 460 nm, and a subpeak is seen around the green-yellow spectral region. The maximum hyperpolarizing response of the RGB cell appears at 400 nm, and it depolarizes over a wide spectral range (460–660 nm). The high sensitivity to blue light of C-type horizontal cells is most characteristic in the dace. The bipolar cells are classified into two groups, non-color-coded cells and color-coded cells, by the spectral properties of the center response. The non-color-coded cells have broad spectral sensitivities, although the spectral maxima are different in the center and the surround in some cells. It is clear from the spectral response curves that there are no large impingements of blue cones in the non-color-coded bipolar cells as compared with the horizontal cells. Spectral properties of the color-coded cells are not yet fully analyzed.     

Design and synthesis of cyclopeptide analogues of the potent histone deacetylase inhibitor FR235222

Various structurally modified analogues of FR235222 (1), a natural tetrapeptide inhibitor of mammalian histone deacetylases, were prepared in a convergent approach. The design of the compounds was aimed to investigate the effect of structural modifications of the tetrapeptide core involved in enzyme binding in order to overcome some synthetic difficulties connected with the natural product 1. The modifications introduced could also help identify key structural features involved in the mechanism of action of these compounds. The prepared molecules were subjected to in vitro pharmacological tests, and their potency was tested on cultured cells. Two of the components of the array were found to be more potent than the parent compound 1 and almost as efficient as trichostatin A (TSA). These results demonstrate that it is possible to synthesize highly active cyclic tetrapeptides using commercially available amino acids (with the exception of 2-amino-8-oxodecanoic acid, Ahoda). The nature of the residue in the second position of the cyclic peptide and the stereochemistry of the Ahoda tail are important for the inhibitory activity of this class of cyclic tetrapeptide analogues.     

Plant Alkaloid Tetrandrine Is a Nuclear Receptor 4A1 Antagonist and Inhibits Panc-1 Cell Growth In Vitro and In Vivo

The orphan nuclear receptor 4A1 (NR4A1) is highly expressed in human pancreatic cancer cells and exerts pro-oncogenic activity. In a previous study, we demonstrated that fangchinoline (FCN), a natural inhibitor of nuclear NR4A1, induces NR4A1-dependent apoptosis in human pancreatic cancer cells. In this study, we evaluated FCN and its structural analogs (berbamine, isotetrandrine, tetrandrine, and tubocurarine) for their inhibitory effects on NR4A1 transactivity, and confirmed that tetrandrine (TTD) showed the highest inhibitory effect in pancreatic cancer cells. Moreover, in a tryptophan fluorescence quenching assay, TTD directly bound to the ligand binding domain (LBD) of NR4A1 with a K_D value of 10.60 μM. Treatment with TTD decreased proliferation and induced apoptosis in Panc-1 human pancreatic cancer cells in part through the reduced expression of the Sp1-dependent anti-apoptotic gene survivin and induction of ROS-mediated endoplasmic reticulum stress, which are the well-known NR4A1-dependent proapoptotic pathways. Furthermore, at a dose of 25 mg/kg/day, TTD reduced tumor growth in an athymic nude mouse xenograft model bearing Panc-1 cells. These data show that TTD is an NR4A1 antagonist and that modulation of the NR4A1-mediated pro-survival pathways is involved in the antitumor effects of TTD.