Yoshinobu Nodasaka

Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals

Osteonectin, an acidic noncollagenous protein of bone and dentin, has affinity to hydroxyapatite crystals. Binding sites to hydroxyapatite of this protein were determined by a proteolytic experiment and an in vitro binding experiment using synthetic peptide analogues. Osteonectin was adsorbed on hydroxyapatite crystals and digested with trypsin. A peptide was left adsorbed on the crystal even after the digestion. The peptide was identified as an amino terminal peptide containing glutamic acid-rich sequences, which have been assumed to be possible hydroxyapatite-binding sites. Poly glutamic acid sequences were synthesized as models of the binding sites. Glu6 peptide was bound to the hydroxyapatite with a dissociation constant of 2.4 microM. Peptides containing fewer glutamic acids had lower affinity to the crystal. Effects of these peptides on in vitro mineralization were examined by a gel system in microtiter plates. The Glu6 peptide had a positive effect on the mineralization in this system, whereas Asp6 peptide had a negative effect. These effects indicate the presence of an interaction between these peptides and mineral crystals.

Pseudomonas alcaliphila sp. nov., a novel facultatively psychrophilic alkaliphile isolated from seawater

Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan. They were Gram-negative, aerobic straight rods with polar flagella. The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C. They produced acid from D-glucose under aerobic conditions. The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA. NaCl was required for growth at pH 10 but was not required at neutral pH. The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol%. The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids. A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas. Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%). On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species. Accordingly, the name Pseudomonas alcaliphila is proposed. The type strain is AL15-21T (= JCM 10630T = IAM 14884T).

Dietzia psychralcaliphila sp. nov., a novel, facultatively psychrophilic alkaliphile that grows on hydrocarbons

A novel, facultatively psychrophilic alkaliphile that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from a drain of a fish product-processing plant. The isolate was an aerobic, non-motile, gram-positive bacterium. The bacterium was catalase-positive and oxidase-negative. The cell wall contained meso-diaminopimelic acid, arabinose and galactose; the glycan moiety of the cell wall contained acetyl residues. The G+C content of the DNA was 69.6 mol %. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was closely related to members of the genus Dietzia (96.1-96.8% similarity). Comparisons of phenotypic and chemotaxonomic characteristics between the isolate and the two known Dietzia species showed that they were very similar. However, the isolate differed from the two known Dietzia species in growth temperature range and certain physiological characteristics. DNA-DNA hybridization revealed that the isolate had 38.4 and 49.7% relatedness, respectively, to Dietzia maris and Dietzia natronolimnaea. On the basis of the physiological and biochemical characteristics, the phylogenetic position as determined by 16S rRNA gene analysis and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Dietzia psychralcaliphila sp. nov. is proposed. The type strain is ILA-1T (= JCM 10987T = IAM14896T = NCIMB 13777T).

Further characterization of interaction between bone sialoprotein (BSP) and collagen.

Bone sialoprotein (BSP) has an affinity to collagen fibrils [25]. A role of carbohydrate chains in the affinity was examined by removing sialic acids of BSP. Neuraminidase treatment of the BSP increased the binding to collagen. Binding sites of BSP on collagen were examined by biochemical and electron-microscopic methods. Purified bovine BSP was labeled with biotin. Collagen α chains or CNBr peptides were separated by electrophoresis and transfered to nitrocellulose membranes. The membranes were incubated with the biotin-labeled BSP, and the bound BSP was visualized with avidin conjugated with alkaline phosphatase. The labeled BSP was preferentially bound to the α 2 chain, and peptides derived from α 2 chain. In another experiment, the labeled BSP was incubated with reconstituted native collagen fibrils. The mixture was put on a copper grid, reacted with avidin conjugated with gold particles, and observed with an electron microscope. The gold particles were seen mainly within hole zones of the fibrils. BSP bound to the α 2 chain within the hole zones may regulate the onset of calcification at hole zones and the cell binding to collagen fibrils.

Attachment of osteoblastic cells to hydroxyapatite crystals by a synthetic peptide (Glu7-Pro-Arg-Gly-Asp-Thr) containing two functional sequences of bone sialoprotein

We investigated activity of bone sialoprotein (BSP) to mediate attachment of cells to hydroxyapatite using a model peptide, Glu7-Pro-Arg-Gly-Asp-Thr, which contains a putative hydroxyapatite-binding site (poly-Glu) and a cell-attachment site. The peptide has affinity to hydroxyapatite with a dissociation constant of 13.5 microM. The peptide affected in vitro mineralization in a gel system, indicating interaction between this peptide and calcium phosphate. The osteoblastic cell line MC3T3-E1 was incubated with hydroxyapatite powder coated with the peptide or proteins. Attachment of the cells was observed on the powder coated with BSP, but not on the powder coated with serum albumin. The cells were attached to the powder coated with the peptide. The cells were flattened on the powder, and pseudopods developed. The attachment of the cells was inhibited by an excessive amount of Gly-Arg-Gly-Asp-Ser peptide. In conclusion, BSP mediated attachment of osteoblastic cells to hydroxyapatite, and this activity could be accomplished only by the poly-Glu sequence and the Arg-Gly-Asp sequence.

Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria.

Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water. The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC). The organisms can utilize PHC and some other biodegradable plastics. The strains grow only under aerobic conditions. Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium. Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively. The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins are not required for growth. The G + C content of genomic DNA is 66.2-66.3 mol%. Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).

Electro-oxidation of CO and methanol on graphite-based platinum electrodes combined with oxide-supported ultrafine gold particles

Abstract Graphite-based Pt electrodes were combined with metal-oxide-supported ultrafine Au particles (diameter C Fe 2 O 3 + Au + Pt electrodes exhibited much higher catalytic activity than the electrodes without Au particles, namely graphite-based Pt ( C Pt ) or graphite-based Pt + α- Fe 2 O 3 ( C Fe 2 O 3 + Pt ) electrodes. The electro-oxidation of CH3OH on the C Fe 2 O 3 + Au + Pt and C Fe 2 O 3 ( Ti-doped ) + Au + Pt electrodes was deactivated to much less than that on either C Pt or C Fe 2 O 3 + Pt electrodes by continuous bubbling of CO. Rather, the apparent activity was increased due to superimposed CO electro-oxidation, particularly at potentials below 500 mV/RHE. The enhancement of activity by oxide-supported Au particles can be attributed to the electro-oxidative removal of CO-like carbonaceous intermediates which were produced by the partial oxidation of CH3OH.

Molybdenum–Vanadium‐Based Molecular Sieves with Microchannels of Seven‐Membered Rings of Corner‐Sharing Metal Oxide Octahedra

Crystalline microporous oxides such as zeolites are indispensable materials in various applications ranging from industrial processes to everyday life, such as catalysts, ion-exchange materials, and molecular sieves. Most of them contain tetrahedrally coordinated metal atoms, but octahedrally coordinated metal centers have recently attracted much attention as building blocks of crystalline microporous metal oxides. Manganese oxides (pyrolusite, hollandite, todorokite, and romanechite) with micropores are the only crystalline porous materials based solely on octahedra (octahedral molecular sieves). These manganese oxides contain microtunnel pores consisting of {MnO6} octahedra that share edges and corners. Here we describe a novel type of octahedral molecular sieve, namely, crystalline orthorhombic Mo3VOx (x = 11.2), in which the microchannel is constructed by seven-membered rings of corner-sharing MO6 (M = Mo or V) octahedra. It is isostructural to orthorhombic MoVNbTeO compounds, which are very active and selective oxidation catalysts for light alkanes. These mixed metal oxides have a layered orthorhombic structure with a slab composed of sixand seven-membered rings of corner-sharing {MO6} octahedra and pentagonal {(M)M5O27} units with a {MO7} pentagonal bipyramid and five edge-sharing {MO6} octahedra, whereM is Mo, V, or Nb. The layered sixand seven-membered rings form channel structures. The Te atom is believed to be located both in the sixand seven-membered rings and block the channel. Recently, we succeeded in preparing an orthorhombic Mo3VOx compound that contains only Mo and V, [6] in which the channel is expected not to be blocked (Figure 1).

Ultrastructure of ceramic-bone interface using hydroxyapatite and β-tricalcium phosphate ceramics and replacement mechanism of β-tricalcium phosphate in bone

Abstract Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are useful for grafting and augmentation of bone tissue. Observation by transmission electron microscopy (TEM) was done to investigate the ultrastructures at the interfaces between the biomaterials and the adjacent tissue, and osteogenesis around the biomaterials in the present study. HA and β-TCP ceramics were used in disk forms which had macropores and micropores, and were implanted between the parietal bone and the cranial periosteum of rats. Specimens were prepared for observation at 4 and 8 weeks postoperatively. The microscopic results indicated that an intervening layer was present on the surface of HA, whereas it was not present on the surface of β-TCP. A characteristic fibrillar structure was observed in the intervening layer between HA and bone under decalcification by HCl. In β-TCP, in reticular structures observed close to the bone tissue by optical microscopy, calcification and sparse collagen fibers were interspersed among the granules of β-TCP. In addition, close to the interface between β-TCP and bone, many osteocytes with numerous processes were present. Some processes were elongated towards the interface. These results revealed the difference in the ultrastructures of the interfaces between HA and β-TCP, and the dissolution mechanism of β-TCP in bone.

Alkalibacterium indicireducens sp. nov., an obligate alkaliphile that reduces indigo dye.

Indigo-reducing, obligately alkaliphilic strains A11T, F11 and F12 were isolated from indigo fermentation liquor obtained from Tokushima Prefecture, Shikoku, Japan. The isolates grew at pH 9.0-12.3, but not at pH 7.0-8.0. The optimum pH range for growth was 9.5-11.5. They were Gram-negative, facultatively anaerobic, rod-shaped strains with peritrichous flagella. The isolates grew in 0-14 % (w/v) NaCl, with optimum growth at 1-11 %. They grew at temperatures of 15-35 degrees C with optimum growth at around 20-30 degrees C. dl-Lactate was the major end product from d-glucose. No quinones were detected. The peptidoglycan type was A4 alpha, l-Lys (l-Orn)-d-Asp. The major cellular fatty acids were C16 : 0, C16 : 17c and C18 : 19c. The DNA G+C contents were 47.0-47.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genus Alkalibacterium. DNA-DNA hybridization revealed low relatedness values between the isolates and the three phylogenetically most closely related species, Alkalibacterium olivapovliticus, Alkalibacterium psychrotolerans and Alkalibacterium iburiense (<41 %). On the basis of phenotypic characteristics, including hydrolysis of cellulose and fermentation of carbohydrates, and chemotaxonomic characteristics, phylogenetic data and DNA-DNA relatedness data, it is concluded that the isolates merit classification as representatives of a novel species of the genus Alkalibacterium, for which the name Alkalibacterium indicireducens sp. nov. is proposed. The type strain of this species is A11T (=JCM 14232T=NCIMB 14253T).