Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1.

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells.

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.

Synthesis of Fluorinated Polymers and Evaluation of Wettability.

Two kinds of fluorinated polymers were synthesized: an acrylate polymer having a fluorinated triethylene glycol as a pendant group (2a) and a fluoroalkyl acrylate polymer (2b). The contact angle of these fluorinated polymers against water, non-fluorinated alcohols and fluorinated alcohols were evaluated. As compared with the fluoroalkyl polymer (2b), fluoroethylene glycol polymer (2a) showed smaller contact angle against water and non-fluorinated alcohols. This supports the proposition that changing the alkyl chain into the ethylene glycol-type chain gave some interaction between etheric oxygen and water or non-fluorinated alcohols. In addition, fluoroalkyl acrylate polymer (2b) showed remarkably low values of critical surface tension.

Effect of aglycon structure on saccharide elongation by cells.

Alkyl N-acetyl-ß-D-glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N-acetyl-ß-D-glucosaminide (GlcNAc-C12), tridecan-7-yl N-acetyl-ß-D-glucosaminide (GlcNAc-2C6), and pentacosan-13-yl N-acetyl-ß-D-glucosaminide (GlcNAc-2C12) primers were synthesized by glycosylation of dodecan-1-ol, tridecan-7-ol, and pentacosan-13-ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc-C12 was elongated to give NeuAc-Gal-GlcNAc-C12. GlcNAc-2C6 was also elongated to afford Gal-GlcNAc-2C6 and NeuAc-Gal-GlcNAc-2C6. On the other hand, GlcNAc-2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5-times by modifying the aglycon structure of GlcNAc from C12 to 2 C6 despite having almost the same number of C-units.

Carbohydrate immobilized on a dendrimer-coated colloidal gold surface for fabrication of a lectin-sensing device based on localized surface plasmon resonance spectroscopy.

Carbohydrate-mediated functions in biological systems have generated considerable interest in recent years. We have developed a device bearing immobilized carbohydrates on a colloidal gold surface and applied this device to the detection of carbohydrate-binding molecules by using localized surface plasmon resonance (LSPR) spectroscopy. The sensing device was constructed by using cyanuric chloride as an amine-linker between an amino residue of a polyamidoamine (PAMAM) dendrimer-coated colloidal gold surface and the amino residue of a 12-aminododecyl glycoside. After optimizing the construction of the device, we characterized its LSPR-based sensing capability. Binding specificity with lectins and linear range responses were obtained with the device. Our LSPR-based sensing device thus provides a label-free, low-cost detection method for use as a laboratory research tool or in medical glycan arrays.

Immobilization of carbohydrate clusters on a quartz crystal microbalance sensor surface.

The immobilization of carbohydrates on gold surfaces is a prerequisite technology for carbohydrate-related studies, including those of carbohydrate-biomolecule interactions. Glycolipid domains in cell membranes, such as lipid rafts, are thought to play an important role in cell biology through their carbohydrate portions. To understand the recognition of glycolipid domains such as receptors for bacterial toxins and viruses, we immobilized clusters of carbohydrates on a gold surface by using polyamidoamine (PAMAM) dendrimers as a scaffold. The PAMAM dendrimers were adsorbed on the gold-coated surface of a quartz crystal microbalance (QCM) sensor and were observed by means of QCM with dissipation (QCM-D). After adsorption of the PAMAM dendrimers, lysoganglioside-GM(1) and 12-aminododecyl-N-acetylglucosaminide (GlcNAc-C12-NH(2)) were immobilized on the amino groups of PAMAM dendrimers by means of an NH(2) cross-linker. Immobilization of the carbohydrates was confirmed by observation of their specific interaction with anti-ganglioside GM(1) antibody or wheat germ agglutinin (WGA). Surfaces with different GlcNAc-C12-NH(2) cluster sizes and densities were prepared by varying the size of the PAMAM dendrimers or the concentration of GlcNAc-C12-NH(2) immobilized on the dendrimers, respectively. Analysis of the binding between the GlcNAc-C12-NH(2)-immobilized surface and WGA revealed that the size of the PAMAM dendrimers influenced the GlcNAc-C12-NH(2)-WGA interaction, with larger dendrimers resulting in higher WGA binding constants.

Influence of passage number on glycosylation of alkyl lactosides by Madin-Darby canine kidney (MDCK) cells.

The cell function on saccharide biosynthesis can be evaluated by employing the saccharide primer method. This study demonstrated that the characteristics of Madin-Darby canine kidney (MDCK) cells changed in relation with passage number when 12-azidododecyl ß-lactoside (Lac-12N(3) primer) was incorporated into MDCK cells and afforded GM3-, GD3-, sialylparagloboside (SPG), and NeuAc-Gal-GlcNAc-Gal-GlcNAc-Lac-type oligosaccharides. By measuring the amount of glycosylated products from relatively early to late passage numbers, results showed that there was an appropriate passage number that optimized oligosaccharide production and that the higher passage number resulted to a decrease in oligosaccharide production. Moreover, results suggested that aside from sialyltransferase, the activity of several kinds of enzymes that control the amount of saccharide production was presumably affected depending upon the biological senescence.

Large scale biosynthesis of ganglioside analogues by RERF-LC-AI cells cultured in HYPERFlask.

The efficient production of ganglioside analogues was accomplished using RERF-LC-AI cells cultured in HYPERFlask (High Yield PERformance Flask). Eight kinds of ganglioside analogues (GM3, GM2, sialylparagloboside, GD3, di-sialylated lacto-N-tetraose, and another three kinds of analogues with intricate structures) were synthesized by the saccharide primer method using lung squamous-cell carcinoma line RERF-LC-AI and 12-azidododecyl ß-lactoside primer. The yield for each analogue obtained using HYPERFlask was higher than yields obtained from 100-mm dishes.

Incorporation of fluorous glycosides to cell membrane and saccharide chain elongation by cellular enzymes.

A series of fluorous-tagged glycosides with different number of fluorine atoms are incorporated into the cells, transported to Golgi, elongated by cellular enzymes, and then released to the culture medium. Fluorine content strongly affects on the affinity for cell membrane and glycosylation. Essentially, the fluorocarbon chain in fluorous compound and the hydrocarbon chain are not miscible. However, the fluorous-tagged glycosides have affinity for cell membrane because of its amphiphilicity. The affinity of fluoro-amphiphilic compound for cell membrane is discussed using critical micelle concentration. The separation of glycosylated products by solvent extraction or fluorous solid phase extraction cartridges is also discussed.

Immobilization of fluorous oligosaccharide recognized by influenza virus on polytetrafluoroethylene filter.

The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galß1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins.

Flow cytometric analysis of lectin-oligosaccharide interactions by using fluorescent oligosaccharide probes.

Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We report here an oligosaccharide binding assay system using tetraphenylethylene derivatives bearing oligosaccharides with aggregation-induced emission (AIE) characteristics. A tetraphenylethylene derivative bearing 6'-sialyllactose (6'SL) bound to microbeads coated with SSA lectin more effectively than RCA120 lectin. Microbeads that bound to fluorescent oligosaccharide probes could be detected by flow cytometric analysis. Tetraphenylethylene derivatives bearing oligosaccharides are useful for flow cytometric analysis of lectin-oligosaccharide interactions.

Novel method for chase analysis of oligosaccharide metabolic error caused by xenobiotics.

Saccharide primers, such as dodecyl beta-lactoside (Lac-C12), are unique artificial precursors of glycolipid synthesis. In culture media supplemented with saccharide primers, they are taken up by the cells in the culture media and glycosylated by cellular glycosyltransferases in the Golgi apparatus to form pseudo-glycolipids. In this study, we examine the effects of various xenobiotics on glycolipid synthesis by implementing a novel method to analyze pseudo-glycolipids, mainly gangliosides, produced by ONS-76 medulloblastoma cells in a culture medium containing various xenobiotics. The ganglioside group of pseudo-glycolipids was effectively purified by using strong anion-exchange cartridges. The production of pseudo-gangliosides was stimulated by N-(n-butyl)deoxygalactonojirimycin (NB-DGJ), but was inhibited by castanospermine, 2-deoxy-2-fluoro-d-galactose, tunicamycin, and brefeldin A. Because the cells in the culture medium are exposed to the saccharide primers and xenobiotics at the same time, and are secreted in the culture medium in their glycosylated form, our method can be used to effectively analyze the direct effects of xenobiotics on ganglioside synthesis.

Development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus.

Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We designed and synthesized a tetraphenylethylene derivative bearing alkyne groups which were used for combination by click chemistry. The new TPE compound bearing alkyne groups was used to synthesize fluorescence oligosaccharide probes which have lactosyl and 6'-sialyllactosyl moieties as ligands. We found that the TPE compounds bearing lactosyl and 6'-sialyllactosyl moieties were useful for detection of RCA120 and SSA lectins, respectively. Moreover, we have shown that TPE-based fluorescent oligosaccharide probe bearing 6'-sialyllactose moiety can be utilized as a "turn-on" fluorescent sensor for detection of influenza virus.

Easy production of a glycolipid analogue using animal cells in culture.

A glycolipid analogue, GM4-type ganglioside, was obtained by a combination of chemical synthesis and biosynthetic processes in animal cells with dodecyl beta-D-galactoside (Gal C12) as primer. The primer was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme-catalyzed glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing methods. The saccharide-primer method is fast, convenient, not requiring expensive enzymes and glycosyl donors, and highly stereoselective.

Structural analysis of glycosphingolipid analogues obtained by the saccharide primer method using CE-ESI-MS.

A glycosphingolipid analogue (12-azidododecyl beta-lactoside) as a saccharide primer has been shown to be useful for the synthesis of oligosaccharide libraries by mammalian cells. In the present study, CE-ESI-MS was employed to elucidate the structure of glycosphingolipid analogues derived from 12-azidododecyl beta-lactoside (Lac-C12N3) by mammalian cells. MDCK cells and COLO201 cells were cultured with Lac-C12N3, and the glycosylated products secreted into the medium were collected and separated into acidic and neutral products by column chromatography. The acidic products could be directly analyzed by CE-ESI-MS, while the neutral products were converted to anionic derivatives via a reaction with propiolic acid. With this method, it was possible to analyze both acidic and neutral products glycosylated by MDCK cells and COLO201 cells at high sensitivity.

Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance.

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.

Purification of gangliosides by liquid-liquid partition chromatography.

A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal partition chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems.

Rapid separation of gangliosides using strong anion exchanger cartridges.

A method utilizing strong anion exchanger cartridges (InertSep SAX) was developed to separate gangliosides. Total lipids extracted from rat brain is able to be rapidly separated into neutral and acidic lipids rapidly. Neutral lipids were passed through the SAX cartridge while acidic lipids adsorbed onto the cartridge and were eluted by chloroform/methanol/4.0 M aqueous ammonium acetate (5:10:1, by volume). Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) were separated individually according to their characteristics by elution with increasing concentration of ammnonium acetate (0 - 4.0 M). The gangliosides yield of this procedure was higher than 95%.

Effects of flow rate on sensitivity and affinity in flow injection biosensor systems studied by 55-MHz wireless quartz crystal microbalance.

In this paper, we developed a 55-MHz wireless-electrodeless quartz crystal microbalance (QCM) and systematically studied the effects of flow rate on the sensitivity to the detection of proteins and on the affinity between biomolecules evaluated by the flow injection system. Brownian motion of proteins in liquid suggests a low probability of meeting, and the convection effect plays an important role in the sensitivity and the affinity in the flow cell injection system. The wireless quartz crystal was isolated in the QCM cell, and flow rates between 50 and 1000 microL/min were used for monitoring binding reactions between human immunoglobulin G and Staphylococcus aureus protein A. The sensitivity was significantly increased as the flow rate increased, while the affinity value remained unchanged. However, the affinity value was affected by the reaction time for a large-concentration analyte, indicating the need of a high-sensitivity biosensor system for accurate evaluation of affinity. The electrode effect on the QCM sensitivity was also theoretically investigated, showing that the electrode significantly deteriorates the QCM sensitivity and makes the Sauerbrey equation invalid.

Purification by centrifugal partition chromatography of amphiphilic compounds, glycolipids and pseudo-glycolipids synthesized by using cells.

Centrifugal partition chromatography (CPC) was applied to separate amphiphilic glycolipids and pseudo-glycolipids synthesized by using cells. Neutral and acidic lipid fractions were isolated by CPC under suitable conditions respectively. Separation of neutral lipid, Gb3-type and Gb4-type oligosaccharide synthesized by using cells, was performed with a two-phase solvent system composed of chloroform-methanol-water at a volume ratio of 5:6:4. On the other hand, separation of acidic lipid, GM3-type oligosaccharide synthesized by using cells, and ganglioside extracted from rat brain were performed with a two-phase solvent system composed of butanol-ethanol-1% acetic acid at a volume ratio of 4:1:5. 8.3mg of Gb3 analogue, 5.1mg of Gb4 analogue, and 19.5mg of GM3 analogue were purified from 3.2l of culture medium obtained by incubation of African green-monkey kidney (Vero) cells with 50 microM n-dodecyl beta-lactoside using CPC.