神澤 信行

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 a 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. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 90A: 811-818, 2009

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 C2C12 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 Ca2+-sensitive proteases, calpains closely take part in post-translational proteolysis.

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both mu- and m- calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain- 1 ( catalytic subunit of mu - 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 C2C12 myoblasts was knocked down with no compensatory overexpression of mu- 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 mu- calpain, and leave us challenging problems in the future.

We have successfully fabricated apatite-fiber scaffolds (AFSs) that enable three-dimensional cell culture. The AFSs possessing large pores of 100-250 mu m and micro pores of about 5 mu m were fabricated by firing the green compacts consisting of the single-crystal apatite fibers and the carbon beads with a size of 150 mu m. In order to enhance the mechanical properties of the AFSs, we have improved the process of AFS fabrication: Collagen gel (type I) solutions were introduced into the pores in the scaffolds; in addition, the resulting apatite/collagen scaffolds were chemically modified by thermally dehydrated cross-linking. Actually, the results of compressive strength tests show that the value of the AFS with chemically cross-linked I-collagen was about twice as high as that of the conventional AFS without I-collagen. We conclude that combination of I-collagen and thermal dehydrated cross-linking is effective for enhancement of the mechanical properties of AFSs.

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 inicroerivironnient 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 microenvironiiient of AFS facilitates cell proliferation and differentiation, and promotes endochondral ossification of bone marrow cells.

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

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.

Vertebrate striated muscle contains the giant elastic protein connectin that maintains the position of the A-band at the center of the sarcomere during repeated muscular contraction and relaxation. Connectin-like molecules may perform conserved functions in vertebrate and invertebrate striated and oblique muscles, although less is known about the structure of invertebrate connectins at present. The protein that maintains such a structure is present not only in vertebrate striated muscle, but also in invertebrate striated and oblique muscle. In the present study, we analyzed the partial primary structure of a 1200K-protein, which is a connectin-like protein that is expressed in Neanthes sp. body wall muscle that is in turn composed of oblique muscle. Antibody screening of a cDNA library of Neanthes sp. body wall muscle identified two different clones. Both clones coded for a sequence predominantly comprised of the four amino acids proline (P), glutamate (E), valine (V) and lysine (K). One clone included a PEVK-like repeat sequence flanked by an Ig domain, while the other clone comprised a distinct 14 amino acid repeat rich in PEVK residues, flanked by a non-repetitive unique sequence. The PEVK region is found in vertebrate connectin and is thought to generate elasticity and be responsible for passive tension of the muscle. The antibodies produced against a portion of each clone both reacted with bands corresponding to 1200 kDa present in Neanthes sp. body wall muscle. Therefore, our results demonstrate that this 1200K-protein is a connectin-like elastic protein and includes specific PEVK-like fragment. We suggest that this 1200K-protein plays a major role in maintaining the structure of oblique muscle in invertebrates.

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. (c) 2005 Elsevier Inc. All rights reserved.

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;I is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

To characterize the biochemical properties of plant annexin, we isolated annexin from Mimosa pudica L. and analyzed the biochemical properties conserved between Mimosa annexin and animal annexins, e.g. the ability to bind phospholipid and F-actin in the presence of calcium. We show that Mimosa annexin is distributed in a wide variety of tissues. Immunoblot analysis also revealed that the amount of annexin is developmentally regulated. To identify novel functions of Mimosa annexin, we examined the pattern of distribution and the regulation of its expression in the pulvinus. The amount of annexin in the pulvinus increased at night and was sensitive to abscisic acid; however, there was no detectable induction of annexin by cold or mechanical stimulus. Annexin distribution in the cell periphery during the daytime was changed to a cytoplasmic distribution at night, indicating that Mimosa annexin may contribute to the nyctinastic movement in the pulvinus.

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. (C) 2004 Elsevier Inc. All rights reserved.

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 11 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, 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. (C) 2003 Elsevier Inc. All rights reserved.

The sessile nature of plants has provided for unique adaptations to their surrounding environments. Over their evolutionary course, plants have survived by changing their functional or structural properties. Animals, being mobile, can change their circumstances to an extent, but plants do not have brains, nervous systems, or muscles. Plants are not, however, static they are able to respond to a variety of environmental stimuli. They perform various macroscopic and microscopic movements in response to intrinsic and extrinsic stimuli.

The biological properties of using two kinds of cells, MC3t3-E1 of an osteoblastic cell line and the rat bone-marrow cell (RBMC) as mesenchymal stem cell model were discussed. The HAp fiber was synthesized via a homogeneous precipitation method using urea. The HAp fiber was mixed with the spherical carbon beads having a diameter of ∼ 150 μm in the mixed solvent. The single HAp phase was present in the S0, S1000, and S2000 scaffolds with porosity of 95% over. The results show that the high porosity and large pore size fulfilled by S2000 could provide an excellent environment for enhancing bone marrow cell activity.

<p>The expression levels of osteoblast differentiation markers and the effect of geometry of porous hydroxyapatite (HAp) fiber scaffold on the differentiation were investigated. The glyceraldehyde-3-phosphate dehydrogenases (GAPDH) mRNA was used as internal control to estimate expression level of differential marker mRNA. An apparent acceleration of the expression was observed for osteoblast differentiation in induced condition. The results show that the materials with various pore sizes effects the three-dimensional apatite fiber scaffold on cell profileration and differentiation.</p>

<p>The effectiveness of hydroxyapatite fibers (HAF) as a scaffold of osteoblastic cells for bone generation was investigated. Three types of HAF with different diameters of pores and porosity were used in the study. The results showed that HAF is degradable and can be eventually replaced by bone tissue in vivo. The results suggested that HAF can be used as an effective scaffold for osteoblast-like cells having capability for bone conduction and that HAF with larger pore sizes can be replaced by newly formed bone tissue in vivo.</p>

The sensitive plant, Mimosa pudica L. closes the leaves and hangs the petiole down when stimulated by touch or heat treatment. This bending of the petiole is caused by rapid shrinking of the lower side of motor organs called the pulvinus. MRI technique indicates that the water in the main pulvinus transfers from the flexor to the extensor during the rapid bending. The actin cytoskeleton in the motor cells plays in important role in the petiole bending. The actin molecules in Mimosa pudica L. is heavily tyrosine-phosphorylated. The bending of Mimosa pudica L. is correlated with reduced actin phosphorylation in the pulvinus.

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.

Achacin is an antibacterial glycoprotein purified from the mucus of the giant snail, Achatina fulica Rrussac, as a Immoral defense factor. We showed that achacin has L-amino acid oxidase activity and can generate cytotoxic H2O2; however, the concentration of H2O2 was not sufficient to kill bacteria. The antibacterial activity of achacin was inhibited by various H2O2 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. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ETA, ETB and ETB2, in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ETA was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ETB was predominantly expressed in the atrium and the left ventricle. ETB2 expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ETA and ETB increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ETA in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.

Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca2+-dependent manner.

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.

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-l (ET-1), an endothelial cell-associated paracrine factor, These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers, It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells, ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the tinting and location of Purkinje fiber differentiation within the embryonic myocardium.

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 Co2+ 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.

We previously reported that novel metallo proteinases, myosinase I and II, which specifically hydrolyzed a myosin heavy chain were purified from squid mantle muscle. In this report, we show the distribution of myosinases in Coleoidea. Spear squid and cuttlefish had myosinase I activity. Japanese common squid had both myosinase I and II activity. In the previous report, we showed that myosinases activity was detected only in extract of Teuthoidea mantle muscle. But in this research, we detected the myosinases activity not only in mantle but also in other tissues other than ovaries from various species of Coleoidea. Each of the livers contained especially high activity. This results suggest that myosinases hydrolyze other substrates.

The texture of fish eggs changes into a unique chewy texture, for example, caviar, during preservation in salt solution. In this work, the changes in fish eggs after preservation in salt solution were investigated. After salt preservation, fish eggs stiffened, and an increase of epsilon-(gamma-glutamyl)lysine (GL) cross-linked products in the chorion fraction was observed. Transglutaminase (TGase) also activated after salt preservation. Therefore, it can be hypothesized that the change of breaking strength after salt preservation was due to the increment of the GL cross-linked products, which was produced by the activation of TGase. Additionally, two kinds of TGase isoforms localized in the chorion fraction of fish egg.