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New computational approach for hydrogen leakage with wall crack propagation problem was conducted by using a hybrid of the coupled particle and Eulerian methods. This computational method provides useful safety information for predicting crack propagation and hydrogen leakage in pressure vessels as an important part of assessing hydrogen as an energy vector. The present computational analysis procedures consisted of two main parts. The first part was crack propagation analysis of a thin square plate, which simulated the wall of a high-pressure hydrogen vessel by using a particle method. The crack propagation was analyzed in high-pressure tank walls under two different types of initial conditions, and in both cases, the direction of crack propagation was freely determined by the direction of the stress field. This confirms the superiority of particle methods for modeling destructive phenomena. After the crack propagation analysis, the particle location and coordinate data of the barrier wall were converted to Euler mesh data. The geometric data of particle location were fitted to Euler numerical space, which was used for simulating high-pressure hydrogen leakage into air at atmospheric pressure. The differences and features of hydrogen diffusion during hydrogen leakage were analyzed for two types of wall data and two types of boundary conditions, as a result, the effect of wall boundaries on the hydrogen concentration distribution was computationally predicted. [ABSTRACT FROM AUTHOR]

Abstract: The clinical efficacy of hepatocyte growth factor (HGF) in tissue repair can be greatly enhanced by high affinity, biocompatible drug carriers that maintain the bioactivity and regulate release at the target site. We produced 0.5–3.0μm fragmin (low molecular weight heparin)/protamine microparticles (F/P MPs) as carriers for the controlled release of HGF. F/P MPs immobilized more than 3μg of HGF per mg of MPs and gradually released the absorbed HGF into the medium with a half-release time of approximately 5days. Compared with HGF alone, HGF-containing F/P MPs substantially enhanced the mitogenic effect of HGF on cultured human microvascular endothelial cells, by prolonging the biological half-life, and its conjugation to F/P MPs protected HGF from heat and proteolytic inactivation. F/P MPs disappeared 8days after subcutaneous injection in mice, suggesting that they are rapidly biodegraded. Furthermore, the number of large (diameter ⩾200μm or containing ⩾100 erythrocytes) and medium (diameter 20–200μm or containing 10–100 erythrocytes) lumen capillaries 8days after injection of HGF-containing F/P MPs was significantly higher than that after injection of HGF or F/P MPs alone. Furthermore, the number of small (diameter ⩽20μm or containing 1–10 erythrocytes) lumen capillaries was significantly higher 4days after injection of HGF-containing F/P MPs. This increased angiogenic activity of HGF in vivo is probably due to both sustained local release and protection against biodegradation by the F/P MPs. Thus, F/P MPs may be useful and safe HGF carriers that facilitate cell proliferation and vascularization at sites of tissue damage. [Copyright &y& Elsevier]

Abstract: The focus of this study is investigation of land subsidence in Semarang city Indonesia with the use of Interferometry Synthetic Aperture Radar (InSAR) of ALOS–PALSAR satellite. We processed 22 ascending SAR images during January 2007 to January 2009 plus two descending SAR images acquired on 6 June 2006 and 17 June 2007. The time series analysis of interferometry was performed by using 12 pairs of interferogram relative to 21 January 2007 and 8 pairs of interferogram relative 24 January 2008. The topographic phase contribution was removed using the 3-arcsec (90m) Shuttle Radar Topography Mission (SRTM), Digital Elevation Model (DEM). We performed precision baseline estimation to vanish the fringes from baseline effect between master and slave data. In order to investigate the contribution of horizontal movement in our analysis, we constructed two interferograms of ascending orbit and descending orbit. The time series results exhibited that the area is subsiding continuously without a significant seasonal effect during January 2007 to January 2009. The land subsidence observed from InSAR data is approximately up to 8cm/year. Three cross sections on image displacement show the extreme land subsidence occurred especially along the coastal area and lowland area where this area is considered as industrial with high-density settlements, consuming a lot of groundwater, and land is changed from agriculture and cultivation purposes to industrial estates and house. Our result also shows a consistency with historical pattern of subsidence measured by leveling data. The results highlight the potential use of InSAR measurements to provide better constraints for land subsidence in Semarang city Indonesia. [Copyright &y& Elsevier]

Abstract: This study focuses on the influence of initiated cracks on the stress distribution within rock samples subjected to indirect tensile loading by traditional Brazilian testing. The numerical analyses show that the stress distribution inside the models is only marginally affected by the friction between the loading platens and the sample. On the other hand, the initiation and propagation of cracks produce a stress field that is very different from that assumed by considering the rock material as continuous, homogeneous, isotropic and elastic. In the models, stress concentrations at the bridges between the cracks were found to reach tensile stresses much higher than the direct tensile strength of the intact rock input in the numerical models. This was due to the development of large stress gradients between the cracks. The analysis of the deformation along the sample diameter perpendicular to the loading direction might enable one to determine the direct tensile strength of the rock. In fact, when the tensile strength is reached, initiation of cracks in the sample induces a sudden increase of the rate of the diametric strains. The direct tensile strength is therefore indicated by the point where the stress–strain curves in the direction perpendicular to the loading diameter depart from linearity. [Copyright &y& Elsevier]

Abstract: Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-β-d-glucopyranoside (GlcNAc-C12) and dodecyl β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including LewisX, sialyl LewisX, polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12. [Copyright &y& Elsevier]

Chitosan has been developed as a polycationic carrier for pDNA in gene delivery system with promising results. On the other hand, the mechanism of the intracellular transport of pDNA/chitosan complexes remains rudimentary yet. In this study, microinjection method was employed to investigate it. The effect of pDNA concentration, post-injection time, cell cycle and stoichiometry of pDNA/chitosan complexes on expression efficiency were investigated. Stability of pDNA complexes in cytosol was also estimated. Furthermore, an atomic force microscope (AFM), a dynamic light scatter and zeta-potential measurement were employed to investigate the physicochemical features of pDNA/chitosan complexes. By the microinjection into nucleus, it was found that complexation of pDNA with chitosan has no negative influence on the expression of pDNA. By the microinjection into cytoplasm, it was shown that chitosan accelerated cell transfection of exogenous genes. Cell division enhances microinjection efficiency. For nuclear import of pDNA/chitosan complexes, optimum N/P ratio was 5, which was well consistent with the results obtained from cellular transfection experiment. Physicochemical features such as the morphology and enzymetic stability of pDNA/chitosan complexes influenced on their nuclear entry.Furthermore, we developed receptor-mediated transfection of the ternary nanoparticles in which pDNA/chitosan particles are coated with hyaluronic acid (HA), which is known to specifically bind CD44. After pDNA/chitosan/HA ternary complexes were prepared, their morphology, particle size and zeta-potential were investigated and it was shown that the ternary complexes were condensed in nano-scale size. Also it was found that pNDA/chitosan/HA complexes were stable in serum, showed protection of pDNA from nuclease attack and low cytotoxicity. In gene transfection experiment, the expression efficiency of pDNA/chitosan complexes in CHO cells that overexpress CD44 on cell surface was enhanced when coated with hyaluronic acid, though the surface charge of the ternary complexes was negative, which is well agreed with the results of cell uptake experiment. Furthermore, Texas-Red labeled chitosan and FITC labeled hyaluronic acid displayed the same cytoplasmic location in CHO cells. These resultus indicated that pDNA/chitosan/ HA ternary naanoparticles were taken up by CHO cells trough receptor-mediated interaction. Cell cycle analysis showed that nuclear entry of the ternary nanoparticles was related with cell division.In this study, to design better gene delivery system that enhances transfection efficiency, we investigated the cell transfection mechanism of pDNA/chitosan complexes. Furthermore, we developed the ternary complexes of pDNA/chitosan coated with hyaluronic acid and clarified their ability as a receptor-mediated gene delivery system.Molecular Therapy (2006) 13, S76–S77; doi: 10.1016/j.ymthe.2006.08.221 [ABSTRACT FROM AUTHOR]

Abstract: Sialyloligosaccharides of glycoproteins and glycosphingolipids play important roles in biological events on cell membranes. GT1b is a ganglioside having a trisialyloligosaccharide and is a receptor for tetanus toxin. In the present study, pentadecapeptide ligands for GT1b were obtained by phage display selection from a random peptide library with the use of a GT1b monolayer. The artificial pentadecapeptides had high affinity for GT1b which tended to increase depending on the number of sialic acids in sialyloligosaccharides. Arg, Ser, and hydrophobic amino acids were found in a consensus motif and may contribute to carbohydrate recognition. The consensus motif of the GT1b-binding peptides was different from that of GM1-, GM2-, GM3-, or GD1a-binding peptides. Peptide ligands for GT1b should be investigated for trisialyloligosaccharide functions and the development of therapeutic agents against trisialyloligosaccharide-related diseases. [Copyright &y& Elsevier]

Abstract: Three ether-linked alkyl 5a-carba-glycopyranosides 1b,d, and 5b, and 5a′-carba-lactoside 7b were examined as potent primers in mouse B16 melanoma cells for their feasibility as building blocks for oligosaccharide biosynthesis. Uptake by B16 cells was first observed for all carba-glycoside primers, and, especially, the 5a-carba-sugar analogues of N-acetyl-β-d-glucosaminide 1b and β-d-glucoside 1d were shown to produce two-to-four-fold larger amounts of glycosylated products than the corresponding true sugar primers 1a and 1c. The carba glycoside uptake by cells resulted in β-galactosylation and subsequent sialylation of the incorporated galactose residues, giving rise to glycosylated products 3b and 3d having similar glycan structures as the ganglioside GM3. According to efficient uptake in cells, in addition to stability of the ether-linked pseudo-reducing ends of the oligosaccharides that formed, the carba glycoside primers have been demonstrated to be versatile building blocks for these biocombinatorial syntheses of glycolipid oligosaccharide mimetics. On the other hand, uptake for 5a-carba-galactopyranoside residue was found to be decreased by one-third for dodecyl 5a-carba-β-d-galactopyranoside 5b. Observation of similar levels for 5a′-carba-β-lactoside 7b under both cellular and cell-free conditions suggested that enzymes are likely to recognize the pyranose oxygen atom. [Copyright &y& Elsevier]

For the purpose of providing biologically stable building blocks for the biocombinatorial synthesis using a living cell, some ether-linked alkyl 5a-carba-β-d-glycoside primers were prepared. The key step of the synthesis was coupling of 1-bromo-n-alkanes with the 1-OH unprotected derivatives of 5a-carba-sugar analogues of d-glucose, d-galactose, and 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), in DMF in the presence of sodium hydride. Alternatively, alkyl carba-lactoside was synthesized by incorporation of a 5a-carba-β-d-galactose residue into the 4-position of dodecyl β-d-glucopyranoside. A strong and specific inhibition of β-galactosidase (Ki 0.67 μM, bovine liver) was found for dodecyl 5a-carba-β-d-galactopyranoside. [Copyright &y& Elsevier]

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N -stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs. [ABSTRACT FROM AUTHOR]

Abstract: Nα -Lauryl-O-(β-d-xylopyranosyl)-l-serinamide (Xyl-Ser-C12) was synthesized as a saccharide primer to obtain oligosaccharides of glycosaminoglycan using the glycan biosynthetic potential of mouse osteosarcoma FBJ-S1 cells and Chinese hamster ovary (CHO) cells. The glycosylated products secreted into the culture medium were collected and analyzed by liquid chromatography–mass spectrometry and glycosidase digestion. The structure of the Xyl-Ser-C12 derivatives was investigated. Several glycosaminoglycan-type oligosaccharides, such as GalNAc-(GlcA-GlcNAc)n-GlcA-Gal-Gal-Xyl-Ser-C12, were detected, and identified as intermediates of the biosynthesis of heparan sulfate glycosaminoglycans. Xyl-Ser-C12 exhibited greater acceptor activity for the glycosylation of glycosaminoglycan-type oligosaccharides than p-nitrophenyl-β-d-xylopyranoside. [Copyright &y& Elsevier]

Abstract: pDNA/chitosan complexes have been investigated as promising non-viral vectors for gene delivery. However, an increase in transfection efficiency and enhancement of physicochemical stability are required for their practical use. In this study, chondroitin sulfate (CS) was employed as a coating agent to increase the stability and transfection efficiency of a pDNA/chitosan complex. The pDNA/chitosan/CS ternary complexes formed with six kinds of CSs having different limiting viscosities (0.2–1.6) and sulfation degrees (5.0–7.0%) showed considerable differences in particle size, surface charge, and morphology. Among them, CS having a medium limiting viscosity (0.5–0.6) and a high sulfation degree (6.9%) showed significant enhancements in cell transfection efficiency. Analyses of cellular uptake and intracellular trafficking revealed that increased cellular uptake via macropinocytosis, together with reduced entry into lysosomes, may explain the promotion of transfection efficiency of ternary complexes. [Copyright &y& Elsevier]

Abstract: The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-restrained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro7–Pro8. Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other hand, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of β- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin. [Copyright &y& Elsevier]

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of β(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing β-galactosidase were used. In the latter, an RGD peptide, the binding site for αvβ3 and αvβ5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with β-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1–1 μg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 μg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection. [Copyright &y& Elsevier]

Mucin-type O -glycosylation of serine or threonine residue in proteins is known to be one of the major post-translational modifications. In this study, two novel alkyl glycosides, N α -lauryl- O -(2-acetamido-2-deoxy-α- d -galactopyranosyl)- l -serineamide (GalNAc-Ser-C12) and N α -lauryl- O -(2-acetamido-2-deoxy-α- d -galactopyranosyl)- l -threonineamide (GalNAc-Thr-C12) were synthesized as saccharide primers to prime mucin-type O -glycan biosynthesis in cells. Upon incubating human gastric cancer MKN45 cells with the saccharide primers, 22 glycosylated products were obtained, and their structures were analyzed using liquid chromatography-mass spectrometry and enzyme digestion. The amounts of glycosylated products were dependent on the amino acid residues in the saccharide primers. For example, in vitro synthesis of T antigen (Galβ1-3GalNAc), fucosyl-T (Fucα1-2Galβ1-3GalNAc), and sialyl-T (NeuAcα2-3Galβ1-3GalNAc) preferred a serine residue, whereas sialyl-Tn (NeuAcα2-6GalNAc) preferred a threonine residue. Furthermore, the glycosylated products derived from GalNAc-Ser/Thr-C12 and Gal-GalNAc-Ser/Thr-C12 using cell-free synthesis showed the same amino acid selectivity as those in the cell experiments. These results indicate that glycosyltransferases involved in the biosynthesis of mucin-type O -glycans distinguish amino acid residues conjugated to GalNAc. The saccharide primers developed in this study might be useful for comparing mucin-type oligosaccharides in cells and constructing oligosaccharide libraries to study cell function. [Display omitted] • Two novel saccharide primers, GalNAc-Ser-C12 and GalNAc-Thr-C12, were synthesized. • O -glycan-type oligosaccharides were obtained by in vitro synthesis using the saccharide primers. • Amino acid selectivity in cellular synthesis of mucin-type O -glycans were observed using the saccharide primers. [ABSTRACT FROM AUTHOR]

Abstract: O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins. [Copyright &y& Elsevier]