Neutralizing the Impact of the Virulence Factor LecA from Pseudomonas aeruginosa on Human Cells with New Glycomimetic Inhibitors.

Bacterial adhesion, biofilm formation and host cell invasion of the ESKAPE pathogen Pseudomonas aeruginosa require the tetravalent lectins LecA and LecB, which are therefore drug targets to fight these infections. Recently, we have reported highly potent divalent galactosides as specific LecA inhibitors. However, they suffered from very low solubility and an intrinsic chemical instability due to two acylhydrazone motifs, which precluded further biological evaluation. Here, we isosterically substituted the acylhydrazones and systematically varied linker identity and length between the two galactosides necessary for LecA binding. The optimized divalent LecA ligands showed improved stability and were up to 1000-fold more soluble. Importantly, these properties now enabled their biological characterization. The lead compound L2 potently inhibited LecA binding to lung epithelial cells, restored wound closure in a scratch assay and reduced the invasiveness of P. aeruginosa into host cells.

Cyanidin 3-O-galactoside: A Natural Compound with Multiple Health Benefits.

Cyanidin 3-O-galactoside (Cy3Gal) is one of the most widespread anthocyanins that positively impacts the health of animals and humans. Since it is available from a wide range of natural sources, such as fruits (apples and berries in particular), substantial studies were performed to investigate its biosynthesis, chemical stability, natural occurrences and content, extraction methods, physiological functions, as well as potential applications. In this review, we focus on presenting the previous studies on the abovementioned aspects of Cy3Gal. As a conclusion, Cy3Gal shares a common biosynthesis pathway and analogous stability with other anthocyanins. Galactosyltransferase utilizing uridine diphosphate galactose (UDP-galactose) and cyanidin as substrates is unique for Cy3Gal biosynthesis. Extraction employing different methods reveals chokeberry as the most practical natural source for mass-production of this compound. The antioxidant properties and other health effects, including anti-inflammatory, anticancer, antidiabetic, anti-toxicity, cardiovascular, and nervous protective capacities, are highlighted in purified Cy3Gal and in its combination with other polyphenols. These unique properties of Cy3Gal are discussed and compared with other anthocyanins with related structure for an in-depth evaluation of its potential value as food additives or health supplement. Emphasis is laid on the description of its physiological functions confirmed via various approaches.

(De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or ß-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that ß-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl ß-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that ß-sheet structures comprise the assembly of the fibrils.

6-Deoxy-6-fluoro galactofuranosides: regioselective glycosylation, unexpected reactivity, and anti-leishmanial activity.

Selective glycosylation of the C-6 fluorinated galactofuranosyl acceptor 2 was studied with four galactofuranosyl donors. It was highlighted that this electron-withdrawing atom strongly impacted the behavior of the acceptor, thus leading to unprecedented glycosylation pathways. Competition between expected glycosylation of 2, ring expansion of this acceptor and furanosylation, and intermolecular aglycon transfer was observed. Further investigation of the fluorinated synthetic compounds showed that the presence of fluorine atom contributed to increase the inhibition of the growth of Leishmania tarentolae, a non-pathogenic strain of Leishmania.

Galectin-2 Has Bactericidal Effects against Helicobacter pylori in a ß-galactoside-Dependent Manner.

Helicobacter pylori is associated with the onset of gastritis, peptic ulcers, and gastric cancer. Galectins are a family of ß-galactoside-binding proteins involved in diverse biological phenomena. Galectin-2 (Gal-2), a member of the galectin family, is predominantly expressed in the gastrointestinal tract. Although some galectin family proteins are involved in immunoreaction, the role of Gal-2 against H. pylori infection remains unclear. In this study, the effects of Gal-2 on H. pylori morphology and survival were examined. Gal-2 induced H. pylori aggregation depending on ß-galactoside and demonstrated a bactericidal effect. Immunohistochemical staining of the gastric tissue indicated that Gal-2 existed in the gastric mucus, as well as mucosa. These results suggested that Gal-2 plays a role in innate immunity against H. pylori infection in gastric mucus.

Effect of Quercetin 3-O-ß-D-Galactopyranoside on the Adipogenic and Osteoblastogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Natural products, especially phenols, are promising therapeutic agents with beneficial effects against aging-related complications such as osteoporosis. This study aimed to investigate the effect of quercetin 3-O-ß-D-galactopyranoside (Q3G), a glycoside of a common bioactive phytochemical quercetin, on osteogenic and adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). hBM-MSCs were induced to differentiate into osteoblasts and adipocytes in the presence or absence of Q3G and the differentiation markers were analyzed to observe the effect. Q3G treatment stimulated the osteoblastogenesis markers: cell proliferation, alkaline phosphatase (ALP) activity and extracellular mineralization. In addition, it upregulated the expression of RUNX2 and osteocalcin protein as osteoblastogenesis regulating transcription factors. Moreover, Q3G treatment increased the activation of osteoblastogenesis-related Wnt and bone morphogenetic protein (BMP) signaling displayed as elevated levels of phosphorylated ß-catenin and Smad1/5 in nuclear fractions of osteo-induced hBM-MSCs. The presence of quercetin in adipo-induced hBM-MSC culture inhibited the adipogenic differentiation depicted as suppressed lipid accumulation and expression of adipogenesis markers such as PPARγ, SREBP1c and C/EBPα. In conclusion, Q3G supplementation stimulated osteoblast differentiation and inhibited adipocyte differentiation in hBM-MSCs via Wnt/BMP and PPARγ pathways, respectively. This study provided useful information of the therapeutic potential of Q3G against osteoporosis mediated via regulation of MSC differentiation.

The Protective Effect of the Polysaccharide Precursor, D-Isofloridoside, from Laurencia undulata on Alcohol-Induced Hepatotoxicity in HepG2 Cells.

Alcoholic liver disease (ALD) threatens human health, so it is imperative that we find ways to prevent or treat it. In recent years, the study of polysaccharides has shown that they have different kinds of bioactivities. Among them are many biological effects that have been attributed to polysaccharide precursors. D-Isofloridoside (DIF) is one of the polysaccharide precursors from the marine red alga Laurencia undulata. This study evaluated the effect of DIF on alcohol-induced oxidative stress in human hepatoma cells (HepG2). As a result, DIF attenuated alcohol-induced cytotoxicity, reduced the amount of intracellular reactive oxygen species (ROS), and effectively reduced alcohol-induced DNA damage in HepG2 cells. In addition, a western blot showed that, after DIF treatment, the expression levels of glutathione (GSH), superoxide dismutase (SOD), and B-cell lymphoma-2 (bcl-2) increased, while the expression levels of γ-glutamyl transferase (GGT), BCL2-associated X (bax), cleaved caspase-3, and mitogen-activated protein kinase (p38 and c-Jun N-terminal kinase ) signal transduction proteins reduced. This showed that DIF may protect cells by reducing the amount of intracellular ROS and inhibiting intracellular oxidative stress and apoptotic processes. Finally, molecular docking demonstrated that DIF can bind to SOD, GGT, B-cell lymphoma-2, and bax proteins. These results indicated that DIF can protect HepG2 cells from alcohol-induced oxidative stress damage, making it an effective potential ingredient in functional foods.

Targeting Bacterial Biofilm: A New LecA Multivalent Ligand with Inhibitory Activity.

Biofilm formation by bacterial pathogens is a hallmark of chronic infections and is associated to increased antibiotic tolerance that makes pathogens difficult to eradicate with conventional antibiotic therapies. Infections caused by Pseudomonas aeruginosa are of great concern, especially for immunocompromised and cystic fibrosis patients. P. aeruginosa lectins LecA and LecB are virulence factors and play a key role in establishing biofilm; therefore, inhibition of the function of these proteins has potential in dismantling the bacterium from the protective biofilm environment and in restoring the activity of antibiotics. Here, we report the NMR characterization of the binding of a galactose-based dendrimer (Gal18) to LecA. Moreover, we demonstrate the activity of the Gal18 molecule in inhibiting P. aeruginosa biofilm formation in vitro.

Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging.

We synthesized new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e6 amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm2). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.

Mechanism of neoagarotetraose protects against intense exercise-induced liver injury based on molecular ecological network analysis.

Here we have explored the effect of neoagarotetraose (NAT) on liver injury caused by intense exercise. Our results showed that NAT treatment obviously decreased liver weight (p < 0.01), improved the liver morphological structure, decreased ALT level (p < 0.05) and endotoxin (LPS) (p < 0.01). In addition, NAT could regulate bile acid profiles in feces and serum of mice, which indicated the potential of liver function, suggesting that NAT was effective to relieve intense exercise-induced liver injury. NAT could regulate the expression of colon genes. NAT tended to alter the microbial composition of mice under intense exercise. We uncovered the network interactions between liver traits and microbial communities in NAT treatment mice. Interestingly, our data indicated that intense exercise-induced liver injury may be related to Clostridiales. In summary, these results demonstrated that NAT relieved liver injury induced by intense exercise may be related to gut microbiota.

Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus.

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.

Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A.

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

Synthesis and structure-activity relationship of theasapogenol galactosides against Magnaporthe oryzae.

Camellia oleifera is expected to provide alternative aglycone to synthesize some saponins similar to that from Schima superba with inhibitory activity against Magnaporthe oryzae. Eight theasapogenol galactosides were synthesized via protection of adjacent hydroxyl groups by a benzylidene for regioselective glycosylation in the multi-hydroxyl sapogenin. Water soluble galactose chain connected far from liposoluble end was a key group in inhibiting the growth of M. oryzea unless theasapogenol was modified by two galactosyl groups or by one galactosyl group and one benzylidene group. The amphoteric characteristics of saponin such as saccharide group number, distance between bipolar groups play an important role in inhibiting mycelium growth of M. oryzae.

Hydrophilic Astragalin Galactoside Induces T Helper Type 1-Mediated Immune Responses via Dendritic Cells.

A flavonoid Astragalin (kaempferol-3-O-ß-d-glucopyranoside, Ast) has several biological activities including anti-oxidant, anti-HIV, and anti-allergic effects. Nonetheless, its insolubility in hydrophilic solvents imposes restrictions on its therapeutic applications. In this study, we investigated the effects of water-soluble astragalin-galactoside (kaempferol-3-O-ß-d-isomaltotrioside, Ast-Gal) on murine bone marrow-derived dendritic cell (DC) maturation and T helper (Th) cell-mediated immune responses. Ast-Gal significantly increased maturation and activation of DCs through the upregulation of surface markers, such as cluster of differentiation (CD)80, CD86, and Major histocompatibility complex (MHC) II in a dose-dependent manner, while Ast had little effects. Additionally, Ast-Gal-treated DCs markedly secreted immune-stimulating cytokines such as interleukin (IL)-1ß, IL-6, and IL-12. Importantly, Ast-Gal strongly increased expression of IL-12, a polarizing cytokine of Th1 cells. In a co-culture system of DCs and CD4⁺ T cells, Ast-Gal-treated DCs preferentially differentiates naïve CD4⁺ T cells into Th1 cells. The addition of neutralizing IL-12 monoclonal antibody (mAb) to cultures of Ast-Gal-treated DCs and CD4⁺ T cells significantly decreased interferon (IFN)-γ production, thereby indicating that Ast-Gal-stimulated DCs enhance the Th1 response through IL-12 production by DCs. Injection with Ast-Gal-treated DCs in mice increased IFN-γ-secreting Th1 cell population. Collectively, these findings indicate that hydrophilically modified astragalin can enhance Th1-mediated immune responses via DCs and point to a possible application of water-soluble astragalin-galactoside as an immune adjuvant.

Curcumin-galactomannoside complex inhibits pathogenesis in Ox-LDL-challenged human peripheral blood mononuclear cells.

Oxidised low-density lipoprotein (ox-LDL) is a pro-atherogenic molecule, which induces inflammatory response and contributes to the pathogenesis of vascular dysfunction to atherosclerosis. The aim of the present study was to explore the anti-inflammatory effect of a novel bioavailable formulation of curcumin as 'curcumagalactomannosides' (CGM) against ox-LDL-induced inflammatory responses in human peripheral blood mononuclear cells (hPBMCs). Curcumagalactomannosides was made from natural curcumin using the soluble dietary fibre (galactomannans) derived from fenugreek seeds (Trigonella foenumgracum) and the hPBMCs were isolated from healthy human volunteers. The cells were cultured in collagen-coated plates at 37 °C and grouped as Group I (Control), Group II (ox-LDL treated) and Group III (ox-LDL + CGM treated). Further analysis of inflammatory markers, reactive oxygen species and mRNA expression levels indicated significantly increased expressions of iNOS, TNF-α, IL-6 and VCAM-1 in ox-LDL-treated group along with the nuclear translocation of NF-κB. Other inflammatory markers such as LOX, PGE2, total COX and lipid peroxidation level were also found to be significantly (p < 0.05) increased upon ox-LDL treatment. The treatment with CGM on the other hand was found to down-regulate and reverse the ox-LDL-induced alterations indicating its potential anti-inflammatory effect on hPBMCs via. NF-κB signalling pathway.

Effector Overlap between the lac and mel Operons of Escherichia coli: Induction of the mel Operon with ß-Galactosides.

The lac (lactose) operon (which processes ß-galactosides) and the mel (melibiose) operon (which processes α-galactosides) of Escherichia coli have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins have been reported over the years. Until now, ß-thiogalactosides like TMG (methyl-ß-d-thiogalactopyranoside) and IPTG (isopropyl-ß-d-thiogalactopyranoside) have not generally been considered to be inducers of the mel operon. The same is true for ß-galactosides such as lactose [ß-d-galactopyranosyl-(1→4)-d-glucose], which is a substrate but is not itself an inducer of the lac operon. This report shows that all three sugars can induce the mel operon significantly when they are accumulated in the cell by Lac permease. Strong induction by ß-thiogalactosides is observed in the presence of Lac permease, and strong induction by lactose (more than 200-fold) is observed in the absence of ß-galactosidase. This finding calls for reevaluation of TMG uptake experiments as assays for Lac permease that were performed with mel+ strains.IMPORTANCE The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. Well-described examples of such interactions are the effector molecules shared by the lac and mel operons. Here, we show that this set has to be extended to include ß-galactosides, which have been, until now, considered not to effect the expression of the mel operon. That they can be inducers of the mel operon as well as the lac operon has not been noted in decades of research because of the Escherichia coli genetic background used in previous studies.

Synthesis and biological properties of galactofuranosyl-containing fluorescent dyes.

Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro.

BACKGROUND: Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). METHODS: Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. RESULTS: Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. CONCLUSIONS: The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.

Different Levels of Skin Whitening Activity among 3,6-Anhydro-l-galactose, Agarooligosaccharides, and Neoagarooligosaccharides.

3,6-Anhydro-l-galactose (AHG), a major monomeric constituent of red macroalgae (Rhodophyta), was recently reported to possess skin whitening activity. Moreover, AHG-containing oligosaccharides, such as agarooligosaccharides (AOSs) and neoagarooligosaccharides (NAOSs), have various physiological activities, including anti-inflammatory, antioxidant, and skin moisturizing effects. In this study, AHG and NAOSs were produced from agarose by enzymatic reactions catalyzed by an endo-type ß-agarase, an exo-type ß-agarase, and a neoagarobiose hydrolase. In a cell proliferation assay, AHG, AOSs, and NAOSs at 12.5, 25, and 50 µg/mL concentrations did not exhibit cytotoxicity toward murine B16 melanoma cells or human epidermal melanocytes. In an in vitro skin whitening activity assay of AHG, AOSs, and NAOSs at 50 µg/mL, AHG showed the highest skin whitening activity in both murine B16 melanoma cells and human epidermal melanocytes; this activity was mediated by the inhibition of melanogenesis. Neoagarotetraose and neoagarohexaose also exhibited in vitro skin whitening activity, whereas neoagarobiose and AOSs with degrees of polymerization of 3 (agarotriose), 5 (agaropentaose), and 7 (agaroheptaose) did not. Therefore, AHG is responsible for the skin whitening activity of agar-derived sugars, and the structural differences among the AHG-containing oligosaccharides may be responsible for their different skin whitening activities.

A dihydrochalcone derivative and further steroidal saponins from Sansevieria trifasciata Prain.

Phytochemical investigation of the aerial parts of Sansevieria trifasciata, one of the most common Dracaenaceae plants, has resulted in the isolation of a new dihydrochalcone derivative named trifasciatine C (1), four previously unreported steroidal saponins as two pairs of inseparable regioisomers: trifasciatosides K/L (2/3), M/N (4/5), together with the known 1,2-(dipalmitoyl)-3-O-ß-D-galactopyranosylglycerol (6), aconitic acid (7), and 1-methyl aconitic acid (8). Their structures were elucidated mainly by extensive spectroscopic analysis (1D and 2D nuclear magnetic resonance) and high-resolution electronspray ionization-mass spectrometry, as well as chemical methods and comparison of their spectral data with those of related compounds. Compounds 2/3 and 4/5 were evaluated for their antiproliferative activity on Hela cells, and no significant effect was observed.