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The need for a selective bacterial recognition method is evident to overcome the global problem of antibiotic resistance. Even though researchers have focused on boronic acid-based nanoprobes that immediately form boronate esters with saccharides at room temperature, the mechanism has not been well studied. We have developed boronic acid-modified poly(amidoamine) (PAMAM) dendrimers with various surface properties to investigate the mechanism of bacterial recognition. The boronic acid-based nanoprobes showed selectivity toward strains, species, or a certain group of bacteria by controlling their surface properties. Our nanoprobes showed selectivity toward Gram-positive bacteria or

Triglyceride (TG) and cholesterol accumulation are known to occur in the liver of rats fed a histidine-excess (5%) diet, but there are few studies reporting histochemical and molecular biological analyses of the rat liver. The aim of this study was to elucidate the molecular basis of this lipid-accumulation mechanism. Lipid accumulations, tissue section images, and gene expression levels were compared in the livers of rats fed a control or histidine-excess diet for 5 wk (n=8/group). Serum levels of TGs, free fatty acids, total cholesterol, high-density lipoprotein cholesterol, glucose, albumin, and the enzyme activities of aspartate aminotransferase and alanine aminotransferase were also analyzed. In the livers of rats fed a histidine-excess diet, histochemical analyses showed what appeared to be a preliminary stage of nonalcoholic fatty liver, characterized by lipid accumulation around the central vein area and minor fibrosis. However, there were no changes in serum TG or free fatty acid levels. Quantitative PCR analyses showed the up-regulation of FAT/CD36, which is related to the uptake of fatty acids into cells, and the downregulation of two apolipoprotein genes, ApoC3 and ApoE. The mRNA levels of PPARγ, LXRα, and AMPKα in the liver were also reduced by excess histidine intake. The results of this study suggest that steatosis caused by excess histidine intake may be the result of an imbalance between lipid transport from the liver and the uptake of free fatty acids into hepatocytes.

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 previously reported that P19.CL6 cells can be cultured in porous hydroxyapatite ceramics prepared by firing green compacts consisting of apatite fibers and spherical carbon beads (150 μm in diameter). Cells cultured for 20 days in an apatite-fiber scaffold (AFS) proliferated and differentiated into cells expressing troponin T, a cardiomyocyte-specific gene, but the expression level was insufficient to support the functional maturation of cells required for biomedical device applications. In this study, we aimed to optimize the internal AFS environment for cardiomyocytes by mixing two sizes (150-and 20-μm) of carbon beads. P19.CL6 cells were cultured in AFS materials comprising different carbon ratios in the presence of alpha-MEM with (AFS+) or without (AFS-) dimethyl sulfoxide (DMSO), and cell growth and gene expression were assessed. We found that AFS(50, 1:1 ratio) is the most suitable scaffold for the proliferation and differentiation of P19.CL6 cells and the addition of DMSO to the culture medium is necessary for differentiation into cardiomyocytes. We also assessed the culture of P19.CL6 cells in AFS in a radial-flow bioreactor for several days.

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 MALDITOF-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.

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 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 β-tricalcium phosphate (β-TCP) phase with anti-washout property (hereafter, β-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 β-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 β-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/β-TCP cement was fabricated by mixing the above-mentioned Ag-HAp/β-TCP powder and 2.5 mass% Na2HPO4 solution at a powder/liquid ratio of 1/0.3 [g/cm3]. 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+ 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 Ca2+-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 or developments are needed for further differentiation.

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 (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 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.

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.

Chelate-setting apatite cement is a novel biomaterial developed as a bone substitute. We previously reported a chelate-setting apatite cement, IP6-HAp, which exhibits anti-tumor activity via apoptotic cell death. However, our preliminary data showed that excess IP6 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 IP6, 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.

Myoglobin from Asian swamp eel Monopterus albus was purified from fish muscle using salt fractionation followed by column chromatography and molecular filtration. The purified Mb of 0.68 mg/g wet weight of muscle was determined for its molecular mass by MALDI-TOF-MS to be 15,525.18 Da. Using isoelectric focusing technique, the purified Mb showed two derivatives with pI of 6.40 and 7.12. Six peptide fragments of this protein identified by LC-MS/MS were homologous to Mbs of sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marlin Makaira nigicans, common carp Cyprinus carpio, and goldfish Carassius auratus. According to the Mb denaturation, the swamp eel Mb had thermal stability higher than walking catfish Clarias batrachus Mb and striped catfish Pangasius hypophthalmus Mb, between 30 and 60 (°)C. For the thermal stability of Mb, the swamp eel Mb showed a biphasic behavior due to the O(2) dissociation and the heme orientation disorder, with the lowest increase in both Kd(f) and Kd(s). The thermal sensitivity of swamp eel Mb was lower than those of the other Mbs for both of fast and slow reaction stages. These results suggest that the swamp eel Mb globin structure is thermally stable, which is consistent with heat-tolerant behavior of the swamp eel particularly in drought habitat.

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.

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.

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 wild-type 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.

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.

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial 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 Ca(2+)-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.

We have successfully synthesized hydroxyapatite fibers via a homogenous precipitation method. Using these hydroxyapatite fibers, we have produced the apatite fiber scaffolds (AFS) with well-controlled pore sizes (porosity above 95%). The AFS is relatively simple to synthesize, and its porosity and pore size are controllable. The usefulness of AFS as a scaffold for bone regeneration was evaluated by (1) seeding and culturing cells in the AFS in vitro, (2) implanting the AFS seeded with cells inside the subcutaneous tissue of mice. The AFS had biocompatibility to support cell adhesion, proliferation, and differentiation. Ectopic bone formation could be formed in the AFS at 12 weeks after implantation into the subcutaneous tissue. Because of its high interpore connection, pore diameters, and porosity, it was believed that AFS was an effective scaffold that provided a three-dimensional cell culture environment. In both in vitro and in vivo environments, the more porous AFS was more advantageous in cell proliferation, cell adhesion, proliferating capacity, robust cell differentiation, ultimately inducing bone ingrowth inside the scaffolds.

The precise role of delta-sarcoglycan (SG) that is constitutively expressed in skeletal muscle cells and may serve for maintaining the sarcolemmal integrity has not been identified. The delta-SG protein is at first among SG complex. To specifically identify the role in C(2)C(12) cells during the myogenesis, we screened several RNA interference (RNAi) candidates at first, and knocked down both levels of the mRNA and protein, employing adenovirus-mediated RNAi. We found no morphological alteration at both myoblast and myotube stages by suppression of delta-SG. The specific knockdown of delta-SG accompanied a concomitant decrease of alpha-, beta-, and gamma-SGs preserving normal levels of each transcript. As for the localization, alpha-, beta-, and gamma-SGs were weakly stained on the cell membrane in delta-SG knockdown cells, whereas each SG in control cell was localized both on the cell membrane and myoplasm abundantly. This enhanced post-translational loss would represent similitude of the progression of cardiomuscular diseases in vitro. Different from cardiac muscle cells, skeletal muscle cell culture without muscle contraction may imply that mechanical stress per se is not primarily involved in the progression of limb-girdle muscular dystrophy. Furthermore, we have observed translocation of calpain-2 to cell membrane in delta-SG knockdown cells, suggesting that Ca(2+)-sensitive proteases, calpains closely take part in post-translational proteolysis.

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both μ- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of μ-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C2C12myoblasts was knocked down with no compensatory overexpression of μ-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca2+level for the full activation plays specific roles in myogenesis, independent of μ-calpain, and leave us challenging problems in the future.

Appropriate culture conditions cause bone marrow stem cells to differentiate into multilineage cells such as adipocytes, chondrocytes, and osteoblasts. One key factor that regulates intercellular signaling and cell differentiation is the extracellular matrix microenvironment. The composition of the extracellular matrix influences cellular functions. In the present study, we investigated the effects of a microenvironment comprising a three-dimensional apatite-fiber scaffold (AFS) that has two kinds of pores (micro- and macro pores) on proliferation and subsequent differentiation of bone marrow stem cells. Morphologic observation revealed that osteoblastic cells in the AFS were distributed primarily in the same location on the fibrous scaffold and formed bridges within micro- and macro pores. We used molecular approaches to evaluate cell proliferation and differentiation in detail. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that culturing bone marrow cells on AFS increases expression of osteocalcin (OC) mRNA compared with culture in a dish. Furthermore, cells cultured in AFS expressed type X collagen (Col X), which is a marker of hypertrophic cartilage. These data suggest that the three-dimensional microenvironment of AFS facilitates cell proliferation and differentiation, and promotes endochondral ossification of bone marrow cells.

Study Design. An experimental study, in which spinal fusion in rats was conducted using a hydroxyapatite fiber mesh (HAM) as a carrier for recombinant human bone morphogenetic protein (rhBMP)-2. Objectives. To study the usefulness of the HAM as a carrier and seek the possibility of clinical application in spinal fusion. Summary of Background Data. Several biomaterials have been used as a carrier for BMP to achieve spine fusion, however, to our knowledge, the most effective carrier has not been established. Methods. In experiment No. 1, HAMs and the controls (commercially available hydroxyapatite ceramic body), loaded with rhBMP-2, were immersed in phosphate-buffered saline to evaluate the time course of the release of rhBMP-2. In experiment No. 2, posterolateral fusion was conducted in rats using HAM and the control loaded with rhBMP-2. The fusion status was evaluated radiologically and histologically after surgery. Results. In experiment No. 1, HAMs released a larger amount of rhBMP-2 for up to 28 days than the controls (49.5% vs 7.8%). In experiment No. 2, the fusion rate was significantly higher in the HAM group (>80%) than in the control group (20%). Dense new bone formed close to the spine, and the HAMs were markedly absorbed compared with the controls. Conclusion. HAM provided more solid fusion mass than the control, suggesting that HAM is an efficient carrier for BMP.

The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement.

Astacin metalloprotease family members function in a wide variety of biologic events, including cell differentiation and morphogenesis during embryonic development and adult tissue differentiation. We previously isolated and characterized an astacin-like squid metalloprotease (ALSM). To elucidate the embryonic expression of ALSM, we performed immunohistochemical analysis with specific antibodies and examined the expression profiles of ALSM isoforms by in situ hybridization analysis. Tissue distribution and expression were also examined in adult spear squid. mRNA expression of ALSM isoforms I and III was first detected in newly hatched squid and was restricted to the liver. No mRNA signals were detected in other tissues even in adult squids. At the protein level, both isoforms were prominent in the liver of embryos and later in digestive organs of adult squid. Both isoforms were also detected in muscle tissues, including mantle and tentacle muscle. Staining for ALSM III was also identified in the iris and in tissues near the eye in squid embryos. However, no reactive bands were detected by immunoblotting of adult squid eyes. Thus, ALSM is initially expressed at the late stage of embryogenesis in spear squid, and expression is restricted to the liver. Thereafter, ALSM isoforms function in various tissues in an isoform-dependent manner.

We studied the mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the chick embryo, which is an organism highly sensitive to TCDD. TCDD was injected into egg yolks prior to embryogenesis, and eggs were incubated for 12 or 18 days. In TCDD-exposed embryos, we observed increased heart wet weight and change in the color of the liver, with abnormal fatty vesicle formation. To determine whether these effects were mediated by the aryl hydrocarbon receptor (AhR), we examined expression levels of AhR, CYP1A4, and CYP1A5. AhR was expressed continuously in the heart and liver during embryogenesis, whereas induction of CYP1A4 and CYP1A5 by TCDD was detected only in the liver. In situ hybridization study of tissue sections revealed induction of CYP1A4 in the abnormal liver tissue in which color change was not observed. To determine whether these different responses to TCDD depended on the cell type, primary cultures of chick hepatocytes and cardiac myocytes were established and 7-ethoxyresorufin-O-deethylase (EROD) activity was measured. Induction of EROD activity following exposure to TCDD was detected in hepatocytes but not in cardiac myocytes. Although the heart is a principal target organ for TCDD toxicity and AhR is expressed throughout embryogenesis, induction of CYP1A was not observed in the chick heart. Thus, we conclude that defects in the heart induced by exposure to TCDD occur via a different pathway than that occurring in the liver.

Achacin, which belongs to the l -amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H2O2. In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H2O2 scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z–VAD–fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H2O2 through the l -amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of l -amino acids by achacin.

Substructure of the myosin rod and its correlation to filament formation is largely based on studies of proteolytic digests and expressed proteins. However, tryptic digestion of myosin always produces polymorphous peptides. Consequently, it is difficult to determine the relation between myosin substructure and filament formation. Similarly, filament formation with recombinant myosin protein is also difficult to interpret because it is never clear whether the recombinant protein folds like the native protein. We recently reported a novel metal protease isolated from squid liver, astacin-like squid metalloprotease (ALSM), which can specifically hydrolyze in vitro myosin heavy chain. In the present study, we examined the solubility properties of the 65-kDa peptide and light meromyosin (LMM) prepared by ALSM isoform II and trypsin digestion, respectively. The 65-kDa peptide is much less soluble than LMM under physiological conditions, even though the length of 65-kDa peptide is shorter than that of LMM. These results suggest that a novel substructure of myosin drives filament assembly.

Achacin is an antibacterial glycoprotein purified from the mucus of the giant snail, Achatina fulica Férussac, as a humoral defense factor. We showed that achacin has L-amino acid oxidase activity and can generate cytotoxic H(2)O(2); however, the concentration of H(2)O(2) was not sufficient to kill bacteria. The antibacterial activity of achacin was inhibited by various H(2)O(2) scavengers. Immunochemical analysis revealed that achacin was preferentially bound to growth-phase bacteria, accounting for the important role in growth-phase-dependent antibacterial activity of achacin. Achacin may act as an important defense molecule against invading bacteria.

The structural stability of fish myosin depends upon species and temperatures of water in which fish live. Primary, secondary, and quaternary structures of myosin heavy chain (MyHC) from three species of fish living at different temperature ranges have been compared with those of rabbit MyHC in order to investigate the differences in stability. Primary structure of MyHC, although being accessible for warm-water and cold-water fish (carp and walleye pollack), was not available in previous for tropical-water fish literature; so in this study primary structure of MyHC of the tropical-water fish amberjack has been determined by cloning and sequencing its cDNA. The MyHC has 1938 amino acid residues (AA), which are almost as much as as those of carp and walleye pollack. The amberjack MyHC is 91–95% homologous with other fish and rabbit MyHCs. There is a discernible difference between animal species with stable myosin rod (amberjack, carp, and rabbit) and walleye pollack with unstable rod. Stable rod species have a high probability of forming coiled-coil around the COOH-terminal end of the rod, while the pollack has a low coiled-coil formation probability. In addition, the average scores of the coiled-coil for myosin rod were rabbit (1.738) > amberjack (1.691) > carp (1.680) > walleye pollack (1.674) which correlated exactly with the observed stability. The results suggest that coiled-coil forming ability, particularly around the COOH-terminal end, directs structural stability of fish myosin rod.

A novel isoform of myosinase was purified to homogeneity from liver of spear squid by sequential chromatographies using SP Sephadex, hydroxylapatite, Zn/Co chelating affinity, and TSK-gel G2000SW columns. Myosinase activity was detected as a single peak of 45-kDa protein by gel filtration. The novel isoform of myosinase specifically hydrolyzed a rabbit skeletal muscle myosin heavy chain into products of 120 and 100 kDa in the presence of Co(2+) ions, and the cleavage site in the myosin heavy chain was quite different from those of two known myosinase isoforms, I and II. Therefore, we named the novel isoform myosinase III. Myosinase III was also distinguishable from myosinase I by its amino-terminal sequence. The sequence showed similarity to an internal sequence of the astacin family.

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 Ca(2+) ion release was observed. These results suggest that dissolution of Ca(2+) 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.

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K + -/NH 4 + -EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain.

Myosin and paramyosin were purified to homogeneity from deep abdominal flexor and extensor, and claw closer and opener muscles of crayfish. Myosins from fast muscle (deep abdominal flexor and extensor) consisted of one species of heavy chain and two species of light chains, Lf21 and Lf18. Myosin from slow muscle (opener) consisted of two species of heavy chains and three species of light chains, Ls31, Ls21 and Ls18. Closer myosin containing both fast and slow types of muscles contained Lf21, Lf18, Ls31, Ls21 and Ls18. The actin-Mg2+-activated, Ca2+-activated, and K+-EDTA-activated ATPase activities of the fast muscle type of myosins were much higher than those of the slow muscle type of myosin. The ATPase activities of closer myosin were in-between. The optimal KCI concentration of the K+-EDTA-activated ATPase activity of crayfish myosins was around 0.4 M as compared to 1 M in rabbit skeletal muscle. Opener myosin was contaminated with a 130 kDa protein. The latter turned out to be a paramyosin isoform. Slow type of muscle including closer muscle contained both 130 kDa and 105 kDa paramyosin isoforms. On the other hand, fast muscle expressed 110 kDa paramyosin isoform.