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Kazuhiko Ishihara - One of the best experts on this subject based on the ideXlab platform.

  • Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts
    Cell and Tissue Research, 2011
    Co-Authors: Eri Ohto-fujita, Tomohiro Konno, Kazuhiko Ishihara, Miho Shimizu, Toshihiro Sugitate, Jun Miyake, Kotaro Yoshimura, Kaori Taniwaki, Takashi Sakurai, Yukio Hasebe
    Abstract:

    We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

  • A bioconjugated phospholipid polymer biointerface with nanometer-scaled structure for highly sensitive immunoassays.
    Methods In Molecular Biology Clifton Nj, 2011
    Co-Authors: Kazuki Nishizawa, Masaki Takai, Kazuhiko Ishihara
    Abstract:

    This method relates to the preparation of a phospholipid polymer platform and the immobilization of an antibody as a bioaffinity ligand onto the platform to construct a biointerface for highly sensitive immunoassays. The specific phospholipid polymer used in this work is poly2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP) (PMBN). The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN surface could prevent nonspecific protein adsorption from an analyte sample without the use of blocking reagents based on the fundamental properties of the MPC units. Furthermore, a nanometer-scaled particle deposition surface is constructed with PMBN by an electrospray deposition method to enhance the sensitivity by increasing the overall surface area of the biointerface.

  • polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high affinity separation of proteins
    Biomacromolecules, 2008
    Co-Authors: Yusuke Goto, Madoka Takai, Tomohiro Konno, Ryosuke Matsuno, Kazuhiko Ishihara
    Abstract:

    Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly(l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobiliz...

  • bioconjugated phospholipid polymer biointerface for enzyme linked immunosorbent assay
    Biomacromolecules, 2008
    Co-Authors: Kazuki Nishizawa, Madoka Takai, Tomohiro Konno, Kazuhiko Ishihara
    Abstract:

    This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer—poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surfac...

  • antibody immobilization to phospholipid polymer layer on gold substrate of quartz crystal microbalance immunosensor
    Colloids and Surfaces B: Biointerfaces, 2007
    Co-Authors: Shigeru Kurosawa, Jongwon Park, Madoka Takai, Kazuhiko Ishihara
    Abstract:

    Abstract To modify gold electrode for immunosensor to construct an artificial cell membrane structure, water-soluble amphiphilic phospholipid polymer, poly[2-methacryloyloxyehtyl phosphorylcholine- co - n -butyl methacrylate- co - p -nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (PMBN)] was applied. The polymer had active ester groups for immobilization of biomolecules and it was converted partially to thiol groups for binding to gold substrates. The partially thiolated PMBN was adsorbed on a gold electrode of quartz crystal microbalance (QCM). Surface characterization of adsorbed PMBN layers was thoroughly investigated with reflectance anisotropy spectroscopy, ellipsometry spectroscopy, dynamic contact angle and X-ray photoelectron spectroscopy measurements. Among several PMBN, having different degree of thiolation, it was concluded that 21.5% thiolated PMBN layer had the most well-ordered phosphorylcholine groups in its outer surface. The proteins adsorption test revealed that the phosphorylcholine group on the outer side of PMBN layers, which was substituted their active ester groups by glycine, showed suppress the non-specific adsorption of proteins, such as bovine serum albumin and γ-globulin. Also, through antigen–antibody binding evaluation, the anti-C-reactive protein antibody immobilized on the PMBN surface worked well and it was confirmed that denaturation of the antibody on the PMBN layers was hardly occurred in spite of 60 days storage at 4 °C. The antibody conjugated phospholipid polymer layer with well-ordered phosphorylcholine group could be outstanding functional membrane for biomedical diagnostic devices without non-specific binding and reduction of immunologic activity of immobilized antibody.

S. Leath - One of the best experts on this subject based on the ideXlab platform.

  • chromosomal location of pm35 a novel aegilops tauschii derived powdery mildew resistance gene introgressed into common wheat triticum aestivum l
    Theoretical and Applied Genetics, 2007
    Co-Authors: J P Murphy, L M Miranda, David Marshall, Christina Cowger, S. Leath
    Abstract:

    A single gene controlling powdery mildew resistance was identified in the North Carolina germplasm line NC96BGTD3 (NCD3) using genetic analysis of F2 derived lines from a NCD3 X Saluda cross. Microsatellite markers linked to this Pm gene were identified and their most likely order was Xcfd7, 10.3 cM, Xgdm43, 8.6 cM, Xcfd26, 11.9 cM, Pm gene. These markers and the Pm gene were assigned to chromosome 5DL by means of Chinese Spring Nullitetrasomic (Nulli5D-tetra5A) and ditelosomic (Dt5DL) lines. A detached leaf test showed a distinctive disease reaction to six pathogen isolates among the NCD3 Pm gene, Pm2 (5DS) and Pm34 (5DL). An allelism test showed independence between Pm34 and the NCD3 Pm gene. Together, the tests provided strong evidence for the presence of a novel Pm gene in NCD3, and this gene was designated Pm35.

  • pm34 a new powdery mildew resistance gene transferred from aegilops tauschii coss to common wheat triticum aestivum l
    Theoretical and Applied Genetics, 2006
    Co-Authors: L M Miranda, J P Murphy, David Marshall, S. Leath
    Abstract:

    Powdery mildew is a major fungal disease in wheat growing areas worldwide. A novel source of resistance to wheat powdery mildew present in the germplasm line NC97BGTD7 was genetically characterized as a monogenic trait in greenhouse and field trials using F2 derived lines from a NC97BGTD7 X Saluda cross. Microsatellite markers were used to map and tag this resistance gene, now designated Pm34. Three co-dominant microsatellite markers linked to Pm34 were identified and their most likely order was established as: Xbarc177-5D, 5.4cM, Pm34, 2.6cM, Xbarc144-5D, 14cM, Xgwm272-5D. These microsatellite markers were previously mapped to the long arm of the 5D chromosome and their positions were confirmed using Chinese Spring nullitetrasomic Nulli5D-tetra5A and ditelosomic Dt5DL lines. Pm2, the only other known Pm gene on chromosome 5D, has been mapped to the short arm and its specificity is different from that of Pm34.

  • Virulence of Blumeria graminis f. sp. tritici on Winter Wheat in the Eastern United States
    Plant disease, 1998
    Co-Authors: A. S. Niewoehner, S. Leath
    Abstract:

    Samples of perithecia of Blumeria graminis f. sp. tritici from senescing wheat leaves were collected by cooperators from 17 states. Ascospores were discharged from perithecia and single-spore isolates were characterized for virulence genes using a differential host series containing 15 known resistance genes. A total of 520 isolates from 17 states were characterized in 1993 and 1994. Virulence frequencies and complexity and racial composition were examined. The data were analyzed for associations among sets of virulence genes and the geographical distribution of phenotypes. Virulence to Pm3c, Pm3f, pm5, Pm6, and Pm7 was present in all states surveyed. Since 1990, virulence to Pm3a has increased in the northeast, and virulence to Pm1, Pm4b, Pm8, and Pm17 has increased across the area surveyed. The resistance genes Pm12 and Pm16 remain highly effective in the southeastern United States. An increase in virulence frequencies and complexity of isolates was observed.

Galen D Stucky - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of highly ordered mesoporous silica materials using sodium silicate and amphiphilic block copolymers
    Chemical Communications, 2000
    Co-Authors: Ji Man Kim, Galen D Stucky
    Abstract:

    A commercially important synthetic approach to highly ordered mesoporous silica materials (SBA-family) with 2-D hexagonal (P6mm), 3-D hexagonal (P63/mmc) and cubic (Im3m and Pm3m) structures, using sodium silicate as the silica source and amphiphilic block copolymers as the structure-directing agents is demonstrated.

  • NONIONIC TRIBLOCK AND STAR DIBLOCK COPOLYMER AND OLIGOMERIC SURFACTANT SYNTHESES OF HIGHLY ORDERED, HYDROTHERMALLY STABLE, MESOPOROUS SILICA STRUCTURES
    Journal of the American Chemical Society, 1998
    Co-Authors: Dongyuan Zhao, Jianglin Feng, Bradley F Chmelka, Galen D Stucky
    Abstract:

    A family of highly ordered mesoporous (20−300 A) silica structures have been synthesized by the use of commercially available nonionic alkyl poly(ethylene oxide) (PEO) oligomeric surfactants and poly(alkylene oxide) block copolymers in acid media. Periodic arrangements of mescoscopically ordered pores with cubic Im3m, cubic Pm3m (or others), 3-d hexagonal (P63/mmc), 2-d hexagonal (p6mm), and lamellar (Lα) symmetries have been prepared. Under acidic conditions at room temperature, the nonionic oligomeric surfactants frequently form cubic or 3-d hexagonal mesoporous silica structures, while the nonionic triblock copolymers tend to form hexagonal (p6mm) mesoporous silica structures. A cubic mesoporous silica structure (SBA-11) with Pm3m diffraction symmetry has been synthesized in the presence of C16H33(OCH2CH2)10OH (C16EO10) surfactant species, while a 3-d hexagonal (P63/mmc) mesoporous silica structure (SBA-12) results when C18EO10 is used. Surfactants with short EO segments tend to form lamellar mesost...

  • oil water interface templating of mesoporous macroscale structures
    Science, 1996
    Co-Authors: S Schacht, Galen D Stucky, Qisheng Huo, I G Voigtmartin, Ferdi Schuth
    Abstract:

    Ordered mesostructured porous silicas that are also macroscopically structured were created by control of the interface on two different length scales simultaneously. Micellar arrays controlled the nanometer-scale assembly, and at the static boundary between an aqueous phase and an organic phase, control was achieved on the micrometer to centimeter scale. Acid-prepared mesostructures of silica were made with the p6, Pm3n, and the P63/mmc structures in the form of porous fibers 50 to 1000 micrometers in length, hollow spheres with diameters of 1 to 100 micrometers, and thin sheets up to 10 centimeters in diameter and about 10 to 500 micrometers in thickness. These results might have implications for technical applications, such as slow drug-release systems or membranes, and in biomineralization, where many processes are also interface-controlled.

  • surfactant control of phases in the synthesis of mesoporous silica based materials
    Chemistry of Materials, 1996
    Co-Authors: And David I Margolese, Galen D Stucky
    Abstract:

    The low-temperature formation of liquid-crystal-like arrays made up of molecular complexes formed between molecular inorganic species and amphiphilic organic molecules is a convenient approach for the synthesis of mesostructure materials. This paper examines how the molecular shapes of covalent organosilanes, quaternary ammonium surfactants, and mixed surfactants in various reaction conditions can be used to synthesize silica-based mesophase configurations, MCM-41 (2d hexagonal, p6m), MCM-48 (cubic Ia3d), MCM-50 (lamellar), SBA-1 (cubic Pm3n), SBA-2 (3d hexagonal P63/mmc), and SBA-3 (hexagonal p6m from acidic synthesis media). The structural function of surfactants in mesophase formation can to a first approximation be related to that of classical surfactants in water or other solvents with parallel roles for organic additives. The effective surfactant ion pair packing parameter, g = V/a0l, remains a useful molecular structure-directing index to characterize the geometry of the mesophase products, and pha...

Beat Keller - One of the best experts on this subject based on the ideXlab platform.

  • single residues in the lrr domain of the wheat pm3a immune receptor can control the strength and the spectrum of the immune response
    Plant Journal, 2020
    Co-Authors: Stefan Lindner, Bettina Keller, Simrat Pal Singh, Zsuzsanna Hasenkamp, Esther Jung, Marion C. Müller, Salim Bourras, Beat Keller
    Abstract:

    The development of improved plant nucleotide-binding, leucine-rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3A2/F2 . PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race-specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.

  • genetic diversity of the pm3 powdery mildew resistance alleles in wheat gene bank accessions as assessed by molecular markers
    Diversity, 2010
    Co-Authors: Navreet K. Bhullar, Michael Mackay, Beat Keller
    Abstract:

    Genetic resources of crop plants are essential for crop breeding. They are conserved in gene banks in form of a large numbers of accessions. These accessions harbor allelic variants of agronomically important genes and molecular tools allow a rapid assessment of this allelic diversity. Here, we have screened a collection of 1005 wheat gene bank accessions for powdery mildew resistance and a molecular characterization for functional alleles at the wheat powdery mildew resistance locus Pm3 was carried out mostly on the resistant accessions. The two analyzed sets of accessions consisted of 733 accessions originating from 20 different countries and 272 landraces originating specifically from Afghanistan. The Pm3 haplotype (indicating the presence of a Pm3-type of gene, susceptible or resistant) was found to be abundantly present in both sets. The accessions with a Pm3 haplotype were further screened for the presence of the functional Pm3a to Pm3g alleles using allele-specific molecular markers. Pm3b and Pm3c were the most frequently found alleles while the other five alleles were detected only in few accessions (Pm3d, Pm3e, Pm3f) or not detected at all (Pm3a, Pm3g). The data further showed that Pm3b is the major source of Pm3-mediated powdery mildew resistance in wheat accessions from Afghanistan. Susceptible allelic variants of Pm3 were found to be widespread in the wheat gene pool. The presented molecular analysis of Pm3 alleles in a diverse set of wheat accessions indicates that several alleles have defined geographical origins. Possibly, the widespread Pm3b and Pm3c alleles evolved relatively early in wheat cultivation, allowing their subsequent diffusion into a broad set of wheat lines.

  • gene specific markers for the wheat gene lr34 yr18 pm38 which confers resistance to multiple fungal pathogens
    Theoretical and Applied Genetics, 2009
    Co-Authors: E S Lagudah, Ravi P Singh, Simon G Krattinger, S A Herrerafoessel, Julio Huertaespino, Wolfgang Spielmeyer, Gina Brownguedira, Liselotte L Selter, Beat Keller
    Abstract:

    The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devastating fungal pathogens leaf rust, stripe rust, and powdery mildew. In previous studies, this broad-spectrum resistance was shown to be controlled by a single gene which encodes a putative ATP-binding cassette transporter. Alleles of resistant and susceptible cultivars differed by only three sequence polymorphisms and the same resistance haplotype was found in the three independent breeding lineages of Lr34/Yr18/Pm38. Hence, we used these conserved sequence polymorphisms as templates to develop diagnostic molecular markers that will assist selection for durable multi-pathogen resistance in breeding programs. Five allele-specific markers (cssfr1-cssfr5) were developed based on a 3 bp deletion in exon 11 of the Lr34-gene, and one marker (cssfr6) was derived from a single nucleotide polymorphism in exon 12. Validation of reference genotypes, well characterized for the presence or absence of the Lr34/Yr18/Pm38 resistance locus, demonstrated perfect diagnostic values for the newly developed markers. By testing the new markers on a larger set of wheat cultivars, a third Lr34 haplotype, not described so far, was discovered in some European winter wheat and spelt material. Some cultivars with uncertain Lr34 status were re-assessed using the newly derived markers. Unambiguous identification of the Lr34 gene aided by the new markers has revealed that some wheat cultivars incorrectly postulated as having Lr34 may possess as yet uncharacterised loci for adult plant leaf and stripe rust resistance.

  • allelic series of four powdery mildew resistance genes at the pm3 locus in hexaploid bread wheat
    Plant Physiology, 2005
    Co-Authors: Payorm Srichumpa, Beat Keller, Susanne Brunner, Nabila Yahiaoui
    Abstract:

    At the Pm3 locus in hexaploid wheat (Triticum aestivum), 10 alleles conferring race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici) are known. A cluster of genes encoding coiled-coil-nucleotide-binding site-leucine-rich repeat proteins spans the Pm3 locus on wheat chromosome 1A, and one member of this gene family has recently been identified as the Pm3b resistance gene. Using molecular markers closely linked to Pm3b, we performed haplotype analysis of 10 lines carrying different Pm3 alleles. All these lines have a conserved genomic region delimited by markers cosegregating with Pm3b and including a structurally conserved Pm3b-like gene. A polymerase chain reaction-based strategy allowed the amplification of one Pm3b-like sequence from lines carrying Pm3a, Pm3d, and Pm3f alleles. These candidate genes for Pm3a, Pm3d, and Pm3f conferred AvrPm3a-, AvrPm3d-, and AvrPm3f-dependent resistance, respectively, to wheat powdery mildew in a single cell transient transformation assay. A high level of amino acid similarity (97.8%) was found between the PM3A, PM3B, PM3D, and PM3F proteins. The coiled-coil domain was 100% conserved, whereas, in the nucleotide binding site region, sequence exchange was detected, indicating intragenic recombination or gene conversion between alleles. All these results indicate that Pm3a, Pm3b, Pm3d, and Pm3f form a true allelic series. The low level of sequence divergence between the four characterized alleles as well as the finding of a conserved Pm3 haplotype are in agreement with the hypothesis of a recent evolution of Pm3-based resistance, suggesting that some or most of the diversity found at the Pm3 locus in modern wheat has evolved after wheat domestication.

  • genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene pm3b from hexaploid wheat
    Plant Journal, 2004
    Co-Authors: Nabila Yahiaoui, Payorm Srichumpa, Robert Dudler, Beat Keller
    Abstract:

    Summary In wheat, race-specific resistance to the fungal pathogen powdery mildew (Blumeria graminis f. sp. tritici) is controlled by the Pm genes. There are 10 alleles conferring resistance at the Pm3 locus (Pm3a to Pm3j) on chromosome 1AS of hexaploid bread wheat (Triticum aestivum L.). The genome of hexaploid wheat has a size of 1.6 × 1010 bp and contains more than 80% of repetitive sequences, making positional cloning difficult. Here, we demonstrate that the combined analysis of genomes from wheat species with different ploidy levels can be exploited for positional cloning in bread wheat. We have mapped the Pm3b gene in hexaploid wheat to a genetic interval of 0.97 centimorgan (cM). The diploid T. monococcum and the tetraploid T. turgidum ssp. durum provided models for the A genome of hexaploid wheat and allowed to establish a physical contig spanning the Pm3 locus. Although the haplotypes at the Pm3 locus differed markedly between the three species, a large resistance gene-like family specific to wheat group 1 chromosomes was consistently found at the Pm3 locus. A candidate gene for Pm3b was identified using partial sequence conservation between resistant line Chul and T. monococcum cv. DV92. A susceptible Pm3b mutant, carrying a single-base pair deletion in the coding region of the candidate gene was isolated. When tested in a single cell transformation assay, the Pm3b candidate gene conferred race-specific resistance to powdery mildew. These results demonstrate that the candidate gene, a member of the coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) type of disease resistance genes, is the Pm3b gene.

K Payer - One of the best experts on this subject based on the ideXlab platform.