Shin-ichi Ogata

Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a β sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation.

The controlled resorption of porous α-tricalcium phosphate using a hydroxypropylcellulose coating

Tricalcium phosphate (TCP) ceramic is known in orthopedics to be a bioresorbable bone substitute. A porous TCP ceramic body also has high potential as a drug delivery system in bony defects. Porous α-TCP ceramic can be easily fabricated using conventional sintering of β-TCP, since α-TCP is the thermodynamically stable phase at temperatures above 1 100 °C. However, the solubility of α-TCP is much higher than that of β-TCP. Therefore, the dissolution of porous α-TCP progresses at a higher rate than bone repair. In the present study, we attempted to reduce the dissolution rate of porous α-TCP by employing an organic polymer coating. We fabricated porous α-TCP ceramic with a continuous 10–50 μm diameter pore structure by sintering a body made from a β-TCP and potato starch slurry. The porous body obtained was coated with hydroxypropylcellulose (HPC), and then subjected to heat treatment. The chemical durability and mechanical properties of the body were examined before and after coating with the HPC. The dissolution of porous α-TCP in buffered solutions was reduced by coating with HPC and drying at 60 °C. The compressive strength of the porous α-TCP was also improved by coating with HPC. The results of in vivo experiments showed that some parts of the porous α-TCP ceramic coated with HPC remained in the canal of the tibia of a rabbit four weeks after implantation, whereas no residual was observed in a non-coated α-TCP ceramic. Coating with HPC was found to be effective for controlling bioresorption and improving the workability of porous α-TCP ceramic. The prepared porous α-TCP ceramic is expected to be useful as a novel material for bone fillers by incorporating it with drugs or osteoinductive factors.

Cellular Uptake and Photocytotoxicity of Glycoconjugated Porphyrins in Hela Cells.

Thirty‐two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)2.2H2O as a template. The Zn2+ ion template strategy improved the yield about three‐fold in the case of meta ‐substituted tetraphenylporphyrins. In addition, free‐base porphyrins were obtained almost quantitatively by demetalation with 4 M HCI. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15, 20‐tetrakis[4‐(β‐D‐arabinopyranosyloxy)phenyl]porphyrin p‐5d) in particular showed 18.5‐fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20‐tetrakis[4‐(β‐D‐glucopyranosyloxy) phenyl]porphyrin (p‐5a), (p‐5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm2. These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS p‐5a p‐5d. These results suggest p‐5d is a good candidate for a PDT drug.

Promotion of neurite outgrowth from fetal hippocampal cells by TNF-alpha receptor 1-derived peptide.

Cytokines such as tumor necrosis factor-α (TNF-α), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand–receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

Preparation of Porous Biphasic Tricalcium Phosphate and Its In Vivo Behavior

Biphasic ceramic consisting of tricalcium phosphate with α- and β -phases (αβ-TCP) is a candidate as biodegradable bone substitutes since its biodegradability may be controlled by the ratio of the phases. In the present study, preparation of porous αβ-TCP body with continuous pores of 10-50 µm in diameter was attempted using additives of Mg, and its in vivo behavior was examined. Powder of β-TCP was mixed with Mg and potato starch to form slurry, followed by loading in polyurethane foam. The sample was fired at 1400°C for 12 hours for sintering process. α-TCP content of the sample decreased with increasing the Mg content, while β-TCP increased. Ceramic body consisting of β-TCP phase was obtained when 1.0 mass% of Mg was added. Porosity of the body decreased with increasing the content of Mg. The αβ-TCP body with 80% porosity was obtained when the content of Mg was 0.1 mass%. The in vivo experiments showed that the rate of degradation of the obtained αβ-TCP was almost same as α-TCP, and much higher than β-TCP.

In Vitro Analysis of Protein Adhesion to Phase Pure Hydroxyapatite and Silicon Substituted Hydroxyapatite

The adhesion of bovine collagen type I, bovine serum albumin, bovine IgG, 1 % and 10 % (v/v) human serum to hydroxyapatite (HA), silicon-substituted hydroxyapatite (Si-HA) and tissue culture plastic were studied. The materials were incubated at 37 °C for 30 minutes, after which the protein solution was removed and analyzed. The adsorbed protein was evaluated by electrophoresis and immunoassay after extraction from the materials. The degree of adhesion was higher for collagen, followed by IgG and albumin on all materials. However there was no difference in the amount of collagen adsorbed onto the surface of each material and this was also the finding with albumin and IgG. These results suggest that the increased bioactivity seen with Si-HA is not due to the degree of protein adhesion, but may possibly be due to changes in the conformation of the bound proteins.

Hydrophobicity parameters (Log P) of glycoconjugated porphyrins for photodynamic therapy evaluated by reversed phase HPLC

Photodynamic therapy (PDT) is one of the most effective treatments for cancer. We synthesized and characterized a series of 16 glycoconjugated porphyrins using a modification of the Lindsey method in the presence of Zn(OAc)2·2H2O as a template. The Zn2+ ion template strategy improved the yield about 3-fold in the case of meta-substituted tetraphenylporphyrins, and free-base porphyrins were obtaind almost quantitatively by demetalation with 4 M HCl. The hydrophobicity parameter (Log P) of these porphyrins was evaluated by reversed phase high-performance liquid chromatography (RP-HPLC). The Log P values ranged from +4.8 to +7.8, so that most of the compounds are amphiphilic. The effect of the glycopyranosyl unit on the hydrophobicity of these compounds is discussed on the basis of the Log P value.

Biodegradation of Porous Alpha-Tricalcium Phosphate Coated with Silk Sericin

Porous a-tricalcium phosphate (a-TCP) ceramics are attractive as a novel bioresorbable material for bone repair, since they can be easily fabricated through conventional sintering of b-TCP at high temperature. However, the solubility of a-TCP is too high to keep its body until the bone defect is repaired completely. Coating of the a-TCP porous body with organic polymer is a way to reduce the degradation rate. In the present study, biodegradation of a-TCP porous body coated with silk sericin was evaluated in vivo. Bone repair at the defect made in rabbit tibia was nearly completed after 4 weeks. Higher density of cortical bone was estimated for a-TCP coated with sericin than for mere a-TCP. The a-TCP porous body coated with sericin is expected as a material that show less degradation than mere a-TCP, and may result in suitable bone repair.

Mutational analysis of the signal for a nuclear localization of proteins which accumulate specifically during meiosis in lily microsporocytes

Abstract LIM5 and LIM13 are novel meiosis-associated genes isolated from Lilium longiflorum. The presence of a hydrophobic N-terminal region predicted from the amino acid sequence has suggested that they function as extracellular structural components. However, both proteins also contain clusters of basic amino acids which may function as nuclear localization signals. To investigate the cellular localization of the protein, we tagged the C-termini of LIM5 and LIM13 with a green fluorescent protein. Transient expression of fusion proteins in onion epidermal cells revealed nuclear localization activity of both proteins. Mutational analysis indicated that amino acid sequences that constitute bipartite-type nuclear localization signals are necessary and sufficient for the intracellular localization of both proteins.

A synthetic peptide corresponding to residues 301-320 of human Wnt-1 promotes PC12 cell adhesion and hippocampal neural stem cell differentiation

Wnt signaling cascades play a crucial role in the maintenance of stem cell niches in many tissues as well as in embryonic patterning and cell-fate determination. Wnt signaling pathways have been well studied; however, the precise binding mechanism of Wnt protein to its receptor has not yet been clarified. Here we show the design and synthesis of seven novel peptide candidates for a receptor-binding site of human Wnt-1 based on its hydrophilicity and beta-turn profiles. Among these Wnt-derived peptides, only WP7, which corresponds to residues 301-320 of human Wnt-1, bound to the soluble receptor for Wnt-1, mouse Frizzled-1/Fc chimera, promoted PC12 cell adherence, increased level of cytosolic beta-catenin in PC12 cells, and induced adhesion and neuronal differentiation of hippocampal neural precursor cells. These results suggest that residues 301-320 of human Wnt-1 is one of the receptor-binding sites and that WP7 may activate the canonical Wnt pathway. When combined with an appropriate matrix, the action of this Wnt-derived peptide, WP7, can be limited to within a location, and therefore could be useful in the regeneration of many tissues, without fear of tumor generation.