Norihiko Minoura

Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks.

We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA-co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA-ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA-co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA-ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA-ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA-ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA-ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.

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.

Development of lipid A-imprinted polymer hydrogels that selectively recognize lipopolysaccharides.

To remove lipopolysaccharide (LPS) from pure water, we developed polymer hydrogels that selectively recognize LPS. A molecular imprinting technique was used to prepare the polymer hydrogels. We prepared the polymer hydrogels with LPS-binding sites by using acryloyllysine and acryloylphenylalanine as functional monomers and used lipid A as a template because it is the biologically active part of LPS and contains two phosphate groups. Co-existence of n-octane during the polymerization process was highly effective in promoting the formation of LPS-accessible sites on the surface of the hydrogels. Both an electrostatic and a hydrophobic interaction between the lipid A portion of LPS and the recognition site of the imprinted hydrogel are necessary for LPS recognition. The adsorption isotherm of LPS to the lipid A-imprinted hydrogels was Langmuir-type; the saturated adsorption capacity and the adsorption constant, calculated by applying an equation for Langmuir-type adsorption isotherms, were 1.0 × 10(-11)mol/cm(2) and 2.5 × 10(5)M(-1), respectively. The imprinted hydrogels selectively recognized toxic LPS in a competition experiment in which two other kinds of LPS with similar chemical structures to that of the LPS of E. coli (toxic LPS) were adsorbed to the lipid A-imprinted hydrogels.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)(3)] shows a new emission peak and the FP value of [Ru(bpy-2Gal)(3)] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)(3)] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (K(d)) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)(3)] to TCF was confirmed by the FP measurement.

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.

Intricate Recognition of Glycolipid-Like Compounds by HIV-1 Envelope Proteins Evaluated with Surface Plasmon Resonance Imaging

Fusion of human immunodeficiency virus (HIV) to the cell membrane occurs by the specific binding of an envelope protein of HIV-1 (gp120 and gp160) and a glycosphingolipid of the cell membrane. In this study, quantitative and array-based affinity evaluation of gp120 and gp160 was performed by surface plasmon resonance (SPR) and the SPR imaging technique using a substrate immobilized with glycolipid-like compounds (Gb3, GM3, and Lac). Quantitative affinity evaluation showed that gp160 specifically bound to Gb3 and Lac compared with GM3, whereas gp120 showed lower binding affinity and specificity. Array-based evaluation showed that gp160 binds to Gb3 more favorably than Lac and GM3.

Development of LC-MS Method for Detection of Mutant Uromodulin Protein

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 → 381, 471, 567, and 679 for GWHWE and m/z 688 → 381, 445, 541, and 653 for GWHC*E) were detected.

Degradation of DNA into 5′-Monodeoxyribonucleotides in the Presence of Mn2+ Ions

DNA is known to be aggregated by metal ions including Mn2+ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn2+ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, as transition element metal ions, were effective as to the degradation into dNMP. Mg2+, Ca2+, Sr2+, and Ba2+, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl−, CH3OO−, and NO3 − were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn2+ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH− ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H1NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.

Detection of mutant uromodulin in transgenic mouse harboring a mutant human UMOD gene.

Uromodulin is the most abundant protein secreted in urine, and the mutated form of the uromodulin gene is associated with uromodulin-associated kidney disease (UAKD). Although uromodulin accumulates in the kidney of UAKD patients, it is unclear whether this is the wildtype or mutant form. In this study, we established a liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS)-based method for the detection of uromodulin mutants, using the C148W mutant as a target molecule. Membrane and cytosolic fractions of kidney samples from transgenic (Tg) mice expressing the C148W uromodulin mutant were shown to contain human uromodulin by western blotting, and mutant uromodulin with the C148W mutant sequence was observed by proteomic and selected reaction monitoring analyses. Our LC-MS/MS-based method is therefore useful for detection of mutant uromodulin that is undetectable by western blotting alone.

Fluorescence Emission and Polarization Analyses for Evaluating Binding of Ruthenium Metalloglycocluster to Lectin and Tetanus Toxin C-Fragment

We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)3] to TCF.