神澤 信行

Ex vivo tissue engineering is an effective therapeutic approach for the treatment of severe cartilage diseases that require tissue replenishment or replacement. This strategy demands scaffolds that are durable enough for long-term cell culture to form artificial tissue. Additionally, such scaffolds must be biocompatible to prevent the transplanted matrix from taking a toll on the patient's body. From the viewpoint of structure and bio-absorbability, a β-tricalcium phosphate (β-TCP) fiber scaffold (βTFS) is expected to serve as a good scaffold for tissue engineering. However, the fragility and high solubility of β-TCP fibers make this matrix unsuitable for long-term cell culture. To solve this problem, we developed an alginate-coated β-TCP fiber scaffold (βTFS-Alg). To assess cell proliferation and differentiation in the presence of βTFS-Alg, we characterized ATDC5 cells, a chondrocyte-like cell line, when grown in this matrix. We found that alginate coated the surface of βTFS fiber and suppressed the elution of Ca2+ from β-TCP fibers. Due to the decreased solubility of βTFS-Alg compared with β-TCP, the former provided an improved scaffold for long-term cell culture. Additionally, we observed superior cell proliferation and upregulation of chondrogenesis marker genes in ATDC5 cells cultured in βTFS-Alg. These results suggest that βTFS-Alg is suitable for application in tissue culture.

Protein adsorption is essential for determining material biocompatibility and promoting adherent cell growth. In this study, we focused on the a-plane structure of hydroxyapatite (HAp). This a-plane structure closely resembles the crystal plane where apatite is exposed in long bones. We conducted protein adsorption experiments using HAp ceramics with a preferred orientation to a-planes (aHAp), employing bovine serum albumin (BSA), lysozyme, and fetal bovine serum (FBS) as protein models to mimic the in vivo environment. Higher zeta potential and contact angle values were found in aHAp than in HAp ceramics fabricated from commercial HAp powder (iHAp). Bradford-quantified protein adsorption revealed BSA adsorption of 212 ng·mm−2 in aHAp and 28.4 ng mm−2 in iHAp. Furthermore, the Bradford-quantified protein adsorption values for FBS were 2.07 μg mm−2 in aHAp and 1.28 µg mm−2 in iHAp. Two-dimensional electrophoresis (2D-PAGE) showed a higher number of protein-derived major spots in aHAp (37 spots) than in iHAp (12 spots). Mass spectrometry analysis of the resulting 2D-PAGE gels revealed proteins adsorbed on aHAp, including secreted frizzled-related protein 3 and vitamin K epoxide reductase complex 1, which are involved in cellular bone differentiation. Overall, these proteins are expected to promote bone differentiation, representing a characteristic property of aHAp.

Flowering locus T (FT) is known to promote flowering in response to photoperiodic conditions and has recently been shown to contribute to other phenomenon, such as diurnal stomatal movement. In legumes, FTs are classified into three subtypes, though the role of each subtype is not well defined. It has been reported that when FT of Lotus japonicus (LjFT) is heterologously expressed in Arabidopsis, LjFT functions as a mobile florigen to promote flowering, similar to Arabidopsis FT (AtFT). In this study, we expressed AtFT in L. japonicus using the SUC2 promoter and showed that heterologous expression of AtFT was able to promote flowering in the plant. We also showed that AtFT expression does not affect stomatal closing nor nyctinastic leaf movement. These findings contribute to our understanding of flower development and have potential application to breeding or plant biotechnology.

We have developed a convenient and selective method for the detection of Gram-positive bacteria using a ditopic poly(amidoamine) (PAMAM) dendrimer probe. The dendrimer that was modified with dipicolylamine (dpa) and phenylboronic acid groups showed selectivity toward Staphylococcus aureus. The ditopic dendrimer system had higher sensitivity and better pH tolerance than the monotopic PAMAM dendrimer probe. We also investigated the mechanisms of various ditopic PAMAM dendrimer probes and found that the selectivity toward Gram-positive bacteria was dependent on a variety of interactions. Supramolecular interactions, such as electrostatic interaction and hydrophobic interaction, per se, did not contribute to the bacterial recognition ability, nor did they improve the selectivity of the ditopic dendrimer system. In contrast, the ditopic PAMAM dendrimer probe that had a phosphate-sensing dpa group and formed a chelate with metal ions showed improved selectivity toward S. aureus. The results suggested that the targeted ditopic PAMAM dendrimer probe showed selectivity toward Gram-positive bacteria. This study is expected to contribute to the elucidation of the interaction between synthetic molecules and bacterial surface. Moreover, our novel method showed potential for the rapid and species-specific recognition of various bacteria.

There is an urgent need to develop a rapid and selective method for the detection of bacteria because delayed diagnosis and the overuse of antibiotics have triggered drug resistance in bacteria. To this end, we prepared boronic acid-modified poly(amidoamine) generation 4 (B-PAMAM(G4)) dendrimer as cross-linking molecules that form aggregates with bacteria. Within 5 min of adding B-PAMAM(G4) dendrimer solution to a bacterial suspension, large aggregates were observed. Interestingly, the aggregate formation with various bacteria was pH-dependent. In basic pH, both Gram-positive and Gram-negative bacteria formed aggregates, but in neutral pH, only Gram-positive bacteria formed aggregates. We revealed that this bacteria-selective aggregation involved the bacterial surface recognition of the phenylboronic acid moiety of B-PAMAM(G4) dendrimer. In addition, we demonstrated that the spherical structure of B-PAMAM(G4) was one of the important factors for the formation of large aggregates. The aggregation was also observed in the presence of ≤10 mM fructose. B-PAMAM(G4) dendrimer is expected to be a powerful tool for the rapid and selective discrimination between Gram-positive and Gram-negative bacteria.

We have developed a new method for Staphylococcus aureus (S. aureus) detection, which employs fluorescent silica nanoparticles (FSiNPs) modified with metal-dipicolylamine complex (M-dpa-HCC). Among the M-dpa-HCC/FSiNP complexes, Cu-dpa-HCC/FSiNP formed large aggregates with S. aureus in 10 min, which were easily observed by the naked eye. The antibacterial activity of Cu-dpa-HCC/FSiNP was also confirmed.

We recently suggested that proline might decrease the suppressive effect of histidine on food intake. Our purpose in the present study was to investigate the influence of proline on the suppressive effect of histidine on food intake and accumulation of body fat. Male Wistar rats were divided into four groups and allowed free access to the following diets for 3 wk: control (C), 5% proline (P), 5% histidine (H), or 5% histidine plus 10% proline (HP) diets. Food intake for 7 d and retroperitoneal fat tissue weight at the end of the experimental period of the HP diet group were greater than those of the H diet group, whereas no significant difference existed between the HP diet group and the C diet group. Our results indicate that proline inhibits the influence of histidine on food intake and accumulation of body fat.

We previously showed enhanced osteoblast differentiation by culturing cells in apatite-fiber scaffold (AFS). The well-developed a-surface of the apatite fibers provides favorable structural features and chemical interactions with the cells, and the scaffold appears to affect cell differentiation. AFS was used here to study its utility for soft-Tissue engineering. An embryonal carcinoma cell line, P19.CL6, was cultured in AFS, and the expression and phosphorylation of a gap-junction protein, connexin 43 (Cx43), during cell proliferation and differentiation was examined. We show that treatment with dimethyl sulfoxide appears to induce a change in the isoform composition of Cx43 under the control condition, but not in AFS. We also show that serum starvation induces the phosphorylation of Ser 368 of Cx43 only in functionally mature cells.

Myoglobin (Mb) was isolated from ordinary muscle of striped snake-head fish. The crude extract was fractionated by salting out and separated using both Sephadex G-75 and DEAE-cellulose column chromatography and molecular filtration, giving at least two protein bands on SDS-PAGE with molecular mass of 15 kDa as major band and trace of 20 kDa compared with those of MALDI-TOF-MS: 15,597.68 Da (major peak) and 23,509.42 Da (trace). Thus, this 15 kDa protein wouldbe the fish Mb which was strongly coupled withother proteins of 23.50 kDa. Spectral characteristics of these purified proteins still exhibited maximum absorption at 280 and 410 nm (the Soret peak). Their tryptophan fluorescence appeared at 330 nm also corresponding with horse heart Mb. The partial peptide sequences of 15 kDa protein obtained from LC-MS/MS with in-gel trypsin digestion were homologous with those of some fish species. In addition, the purified Mb was also determined for its partial peptide sequence by digestion with Lys-C endoproteinase and analysis of amino acid sequence using Edman degradation, resulted in only some amino acid residues are identical to Mb from other fish species. It is noted that differences in their amino acid sequences are attributed among fish Mb species of which playing distinct roles of both structural rigidity and functional property.

Primary structure of myoglobin (Mb) from ordinary muscle of striped snake-head fish (Ophicephalus striatus) was studied. Sequence analysis of cloned cDNA revealed two lengths of nucleotide sequences different in 3' untranslated regions, MbI gene and MbII gene. In accordance to amino acid coding region, both genes encoded protein with 145 amino acid residues which were different in two amino acid residues at position 114 and 117, indicating Mb isoforms. They are very close to those of various species of fish Mbs and very identical to some marine Mbs. There are amino acid differences among the fish Mbs and other Mbs that occur in the highly conserved regions in fish species. The replacement of alanine with serine 59 in fish species, adjacent to histidine 60 may contribute to the reversible binding of oxygen. Lysine was replaced by asparagine at position 93 which directly binds to the heme. The fish Mbs contain two cysteine residues. One of cysteine residue locates at the terminal of the polypeptide chain which may possibly contribute to internal disulfide interaction and thus forms tightly folded structure. It is implied that the fish Mb might be a novel protein with strong intramolecular bonding.

Similar to other glucocorticoids, dexamethasone (DEX) induces osteoblast differentiation. At high concentrations, glucocorticoids may induce osteoporosis as a side effect. However, the exact mechanism of these two opposing effects has not been elucidated. To understand the mechanism of DEX-induced osteoblast differentiation, we developed a real-time osteoblast differentiation detection system using dual labeling of cells with fluorescent proteins. The promoter sequences of type I collagen and osteocalcin were ligated with mCherry and green fluorescent protein (GFP), respectively. Type I collagen is an early marker of osteoblast differentiation, and osteocalcin is a terminal differentiation marker. We investigated the effects of DEX on cell proliferation and differentiation using cells transformed with both constructs. Low DEX concentrations (&lt 10 μM) induced calcification, as determined by alizarin-red staining, whereas calcification was inhibited at higher concentrations (&gt 100 μM). Consistent with these results, mCherry-associated red fluorescence as an early marker was evident under both conditions, whereas green fluorescence associated with terminal differentiation was evident only at lower DEX concentrations. The level of green fluorescence diminished in a DEX-concentration-dependent manner. Thus, DEX does not inhibit the early stages of osteoblast differentiation but instead inhibits terminal differentiation.

We have previously synthesized silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique. On the other hand, we have successfully fabricated novel calcium-phosphate cements (CPCs) composed of mainly |3-tricalcium phosphate (|3-TCP) phase with anti-washout property (hereafter, |3-TCP cement), which was set on the basis of chelate-bonding ability of inositol phosphate (IP6). In this study, we developed novel CPCs with both anti-bacterial and anti-washout properties by adding the Ag-HAp powder into the above |3-TCP cements, and examined their anti-bacterial property and cytotoxicity. The Ag-HAp powders with Ag contents of 0, 2, and 5 mol% as a nominal composition were synthesized by an USSP technique. The raw powder for β-TCP cement was prepared by ball-milling the commercially-available |3-TCP powder in the IP6 solution. The Ag-HAp/β-TCP powders were prepared by mixing Ag-HAp powder and β-TCP cement powder at a ratio of 25:75 in mass. The Ag-HAp/|3-TCP cement was fabricated by mixing the above-mentioned Ag-HAp/β-TCP powder and 2.5 mass% Na&lt inf&gt 2&lt /inf&gt HP0&lt inf&gt 4&lt /inf&gt solution at a powder/liquid ratio of 1/0.3 [g/cm&lt inf&gt 3&lt /inf&gt ]. The anti-bacterial property of resulting cements was evaluated using Staphylococcus aureus by biofilm formation test. The Ag-HAp/β-TCP cements containing 2 and 5 mol% Ag showed strong anti-bacterial property among examined specimens. Furthermore, the cytotoxicity of Ag&lt inf&gt +&lt /inf&gt ion eluted from these cements was also examined using osteoblastic MC3T3-E1 cells and Transwell® kit. The relative cell viability cultured on each Ag-containing cement specimen was over 80 %, compared with the control (polystyrene plate). These results demonstrate that the present Ag-HAp/β-TCP cements containing 2 mol% Ag are promising one of the candidates as CPCs with both anti-bacterial property and biocompatibility.

In the present study, we used an apatite-fiber scaffold (AFS) to culture P19.CL6 cells three-dimensionally. AFS was originally developed to use for the bone substitute and has high porosity and complex pore structure. The highly porous structure of AFS suggests it may be compatible for the in vitro reorganization of soft tissue. We previously showed the formation of small cell aggregates in AFS, and that culture in AFS increased the expression of cardiac-specific gene markers without the need for any inducing agent such as DMSO. However, it is difficult to evaluate the physiological function of cells in three-dimensional culture. In this study, we transformed P19.CL6 cells with the pTnnt2::GCaMP5G vector and observed the cells by fluorescence microscopy. pTnnt2::GCaMP5G consists of green fluorescent protein (GFP) fused with the Ca&lt inf&gt 2+&lt /inf&gt -sensitive domain of calmodulin its expression is driven by a cardiomyocyte-specific promoter. We observed that the blinking of green fluorescence was synchronized to the beating of cardiomyocytes when the P19.CL6 cells were cultured in a dish, but blinking was not observed when the cells were cultured in AFS, even after 16 days. The expression of connexin 43 (Cx43) and enhanced Green Fluorescence Protein (EGFP) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cx43 is a gap junction protein expressed in cardiomyocytes that mediates cell-to-cell coupling. Although the expression of Cx43 and EGFP in transformed cells cultured in AFS was evident, fluorescence blinking of the cells was not observed. The results demonstrate that P19.CL6 cells cultured in AFS rapidly differentiated into early stage cardiomyocytes however, additional modifications ordevelopments are needed for further differentiation.

Hydroxyapatite (HAp), with its high biocompatibility and osteoconductivity, readily absorbs proteins, amino acids and other substances, which in turn favor the adsorption and colonization of bacteria. To prevent bacterial growth and biofilm formation on HAp discs, silver-containing (1-20 mol%) HAp (Ag-HAp) powders were synthesized using an ultrasonic spray pyrolysis (USSP) technique. The X-ray diffraction (XRD) peaks were very broad, indicating low crystallinity, and this induced the release of Ag+ ions from Ag-HAp powders. In addition, a gradual increase in Ca2+ ion release was observed. These results suggest that dissolution of Ca2+ ion in Ag-HAp triggered the release of Ag+ ions. The antimicrobial efficacy of Ag-HAp disc was tested against Staphylococcus aureus. Samples with Ag contents of more than 5 mol%were found to be highly effective against bacterial colonization and biofilm formation in vitro. In vivo antibacterial tests using bioluminescent strains also showed reductions in the viability of bacteria with Ag-HAp (5 mol%) discs. Biocompatibility tests using a modified Transwell® insert method showed that Ag-HAp (5 mol%) discs have negative effects on osteoblast proliferation. These results indicate that Ag-HAp (5 mol%) has effective antibacterial activity and good biocompatibility both in vitro and in vivo together with good biocompatibility, thus confirming its utility as a bactericidal material. © 2013 Elsevier B.V. All rights reserved.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are pluripotent progenitor cells that can regenerate different skeletal tissues in response to environmental signals. Scaffolds play a critical role in tissue engineering, and their microstructure is essential for inductive bone formation. In the present study, we have used highly porous, structurally stable three-dimensional apatite-fiber scaffolds (AFSs) and investigated their ability to support cell proliferation and differentiation. BMSCs in/on AFSs were proliferated in a three-dimensional manner when both micro-and macro-pores were present. The characteristic structure of the AFS enhanced calcification, production of extracellular matrix (ECM), and osteogenic differentiation, especially endochondral ossification. Furthermore, BMSCs cultured in/on AFSs did not undergo adipogenesis and selectively underwent osteogenic differentiation. These results suggest that AFSs provide a suitable environment for bone regeneration and have potential applications for tissue engineering. (C)2013 The Ceramic Society of Japan. All rights reserved.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are pluripotent progenitor cells that can regenerate different skeletal tissues in response to environmental signals. Scaffolds play a critical role in tissue engineering, and their microstructure is essential for inductive bone formation. In the present study, we have used highly porous, structurally stable three-dimensional apatite-fiber scaffolds (AFSs) and investigated their ability to support cell proliferation and differentiation. BMSCs in/on AFSs were proliferated in a three-dimensional manner when both micro- and macro-pores were present. The characteristic structure of the AFS enhanced calcification, production of extracellular matrix (ECM), and osteogenic differentiation, especially endochondral ossification. Furthermore, BMSCs cultured in/on AFSs did not undergo adipogenesis and selectively underwent osteogenic differentiation. These results suggest that AFSs provide a suitable environment for bone regeneration and have potential applications for tissue engineering. © 2013 The Ceramic Society of Japan. All rights reserved.

Two apyrases having different substrate specificity, MP67 and MpAPY2, are present in Mimosa pudica. The substrate specificity of MP67 is quite high against ADP, and is distinct from any other apyrase. This might be attributed to the nucleotide binding motif (DXG) in apyrase conserved region 1. We performed a single amino acid substitution at position X in the motif. The ratio of the velocity of ATP/ADP hydrolysis was higher (approximately 1) for the S63A-MP67 mutant than for wild type-MP67 (0.19). Binding affinity for ADP of A75S-MpAPY2 mutant was increased to a level higher than that of the wild type MpAPY2. Thus, the residue at position X in the DXG motif plays an important role in determining nucleotide preference. © 2013 Landes Bioscience.

Chelate-setting apatite cement is a novel biomaterial developed as a bone substitute. We previously reported a chelate-setting apatite cement, 26-HAp, which exhibits anti-tumor activity via apoptotic cell death. However, our preliminary data showed that excess 26 arrests osteoblast growth. We found that a high, transient amount of IP6 was released from the cement. We therefore hypothesized that a high performance cement specific for tumor cells can be developed by controlling the release of IP6 from the cement. To validate this, we used a murine calvarial osteoblast cell line (MC3T3-E1) and a human osteosarcoma cell line (HOS). Culturing HOS or MC3T3-E1 in medium containing various concentrations of IP6 more effectively arrested the growth of HOS than that of MC3T3-E1. Although the proliferation of osteoblasts was suppressed at early growth stages in response to the release of IP6 from the cements, there was no difference in the number of cells after a prolonged culture period. In contrast, osteosarcoma cell growth remained suppressed even after a prolonged culture period. To better understand why these two cell types respond differently to 26, we investigated cell viability by measuring the ratio of living and dead cells. Our findings suggest that this novel bone graft cement will find unique uses due the different sensitivity of tumor cells and osteoblasts towards IP6.

We have developed a chelate-setting apatite cement. Synthesized hydroxyapatite (HAp) powders surface-modified with inositol hexaphosphate (IP6-HAp powder) were set by chelate-bonding with inositol hexaphosphate (IP6). Our aim is to fabricate IP6-HAp cement with anti-bacterial activity by adding lactoferrin (LF). It is known that LF has both anti-bacterial and osteoinductive activity. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were used to examine the effect of LF on biofilm formation and localization of living and dead cells. In addition, the cell viability of MC3T3-E1 osteoblastic cells was determined. Our results show that the anti-bacterial activity of LF is not due to a bactericidal effect but to the inhibition of bacterial adhesion to surfaces. Furthermore, LF cement did not affect cell proliferation. Thus, LF cement is a candidate for bifunctional biomaterials having both anti-bacterial and osteo-conductive activity.

Heart disease is the second most common cause of mortality in Japan. Most cases of late stage heart failure can only be effectively treated by a heart transplant. Cardiac tissue engineering is emerging both as a new approach for improving the treatment of heart failure and for developing new cardiac drugs. Apatite-fiber scaffold (AFS) was originally designed as a substitute material for bone. AFS contains two sizes of pores and is appropriate for the three dimensional proliferation and differentiation of osteoblasts. To establish engineered heart tissue, a pluripotent embryonal carcinoma cell line, P19.CL6, was cultured in AFS. P19.CL6 cells seeded into AFS proliferated well. Generally, cardiac differentiation of P19.CL6 cells is induced by treating suspension-cultured cells with dimethyl sulfoxide (DMSO), after which the cells form spheroids. However, our results showed that P19.CL6 cells cultured in AFS differentiated into myocytes without forming spheroidal aggregates, and could be cultured for at least one month. Thus, we conclude that AFS is a good candidate as a scaffold for cardiac tissue engineering.

The anti-tumor activity of hydroxyapatite (HAp) cements, which had been developed using a novel setting mechanism termed chelate bonding, against the human osteosarcoma cell line (HOS) and rat bone marrow stromal cells (BMSC) was examined. We aimed to understand the mechanism of the anti-tumor activity of the cement, thereby facilitating improvement of its biological activity. HAp powders were surface-modified with three different concentrations of inositol hexaphosphate (IP6), which were then used to fabricate three different IP6-HAp cements. The amount of IP6 that was bound to the HAp powder, and the amount that was released from the HAp cement, was measured for each sample. Approximately 1/200 to 1/1600 of the bound IP6 was released into the culture medium by day 4. Surface-modification of HAp with high concentration of IP6 inhibits the proliferation of both HOS cells and BMSCs, and appears to induce their apoptotic cell death. HOS cells were slightly more sensitive to IP6 than BMSCs. Thus, novel, chelate-bonded HAp cements are a candidate bone substitution material that exhibit anti-tumor activity. © (2012) Trans Tech Publications.

We prepared silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique and evaluated their antibacterial activity. Ag-HAp powders with Ag contents of 0, 1, 2, 5, and 20 mol% as nominal composition were prepared by the above USSP technique. The droplets of starting solutions were dried at 300 °C and then pyrolyzed at 850 °C to prepare the Ag-HAp powders. Ag-HAp powders showed a HAp single phase and were composed of spherical particles with a diameter of 0.5 - 3.0 μm. To evaluate the antibacterial activity of resulting powders, colony count method was performed using Staphylococcus aureus. Ag-HAp powders with the contents of Ag more than 5 mol% showed good antibacterial activity. © (2012) Trans Tech Publications.

Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wildtype GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.

To understand better the host defense mechanisms of mollusks against pathogens, we examined the antimicrobial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca2+-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.

To apply biodegradable calcium-phosphate microspheres as a carrier for drug delivery system (DDS), the hollow HAp and TCP microspheres were prepared by the ultrasonic spray-pyrolysis (USSP) technique. In the present work, we prepared the hollow bioceramics microspheres with nano-size pores on the surface via a salt-assisted USSP technique on the basis of previous findings. Diameters of the microspheres were in the range of 0.5 to 3.0 mu m, and the nano-pores with the size of similar to 50 nm were present on the surface of the microspheres. The loaded amount of the drug depended on the specific surface area (SSA) value of the microspheres; the washed powders derived from the NaCl concentration of 1.00 mol.dm(-3) showed the greatest amounts of released drug among examined sample powders. The microspheres with nano-size pores on the surface showed a two-step drug release behavior.

A variety of calcium phosphates have been used for bone tissue-engineering applications. We developed porous hydroxyapatite (HAP) ceramics by firing green compacts consisting of spherical carbon beads and HAP fiber. The apatite-fiber scaffold (AFS) forms a three-dimensional network of fibers with two different pore sizes (micro- and macropores). In this study, we investigated cell distribution and fine cell structure in AFS by confocal laser scanning microscopy. Osteoblastic cells were permeated homogenously throughout the scaffold under static culture conditions and grew three-dimensionally in macropores of AFS. Cells penetrated into micropores when they were capable of cell-cell formations. Cell proliferation and differentiation were also evaluated by biochemical and molecular biological approaches. The expression levels of early-phase osteogenic genes in AFS increased immediately, and those of middle-phase genes were maintained during the 2-week study period. Furthermore, the expression of late-phase markers increased gradually during the incubation period. These data indicate that macropores provide sufficient space for cell growth and proliferation and that micropores facilitate cell differentiation via cell-cell networks. This study provides evidence for the effectiveness of three-dimensional culture systems comprising AFS, which mimics the microenvironment of bone cells. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 94A: 937944, 2010.