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Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both [micro]- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of [micro]-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 [C.sub.2][C.sub.12] myoblasts was knocked down with no compensatory overexpression of [micro]-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 [Ca.sup.2+] level for the full activation plays specific roles in myogenesis, independent of [micro]-calpain, and leave us challenging problems in the future. RNA interference; muscle cell development; fusion; adenovirus vector

A study of the effects of heat on the structure of natural actomyosin taken from a muscle of a live carp used biophysical and biochemical methods to reveal the thermal denaturation process of actomyosin. At 30 degrees centigrade, actomyosin molecules start to unfold, allowing hydrophobic amino acid residues and reactive sulfhydryls to surface, and begin to form aggregates as a result of hydrophobic interactions and disulfide bonds. Above 40 degrees centigrade, myosin dissociates from the actin filaments, which start to separate. At 60 degrees centigrade and above, the actomyosin molecules group into larger aggregates, a portion of which is insoluble.

The setting response of fish meat paste starts with unfolding of the alpha-helix of actomyosin (AM) in fish. Analysis of circular dichroism of AM from eight different species of fish indicates that the amount of aplha-helicity differs among various species. Setting involves transformation from solution to gel of salted fish meat paste when kept at low temperatures. The alpha-helicity of the AM decreases during incubation at 30 degrees or 40 degrees Centigrade.

Author(s): Kazuhisa Kameyama [1]; Yoshiro Kishi [2]; Masanori Yoshimura [1]; Nobuyuki Kanzawa [1]; Masazumi Sameshima [2]; Takahide Tsuchiya (corresponding author) [1] The phosphorylation of the amino acid tyrosine in animal [...]

The article discusses the isolation of high molecular weight from the mucus of the giant African snail Achatina fulica. It examines the anti-microbial activity of mucus from the said African snail. It notes that the lectin isolated from the mucus of the snail contributes to its immunity. It cites that lectin was conveyed in the tissues of the mantle collar.

Purpose: We aimed to determine the effects of human umbilical cord blood (UCB)-derived natural killer T (NKT) cells as immunological effectors against hematological malignancies, as well as auto- or allo-dendritic cells (DCs) or EB transformed cell lines (EBCLs). Materials: TCRVα24+ Vβ11+ UCB- or PB-NKT cells were isolated by sorting and activated by α-galactosylceramide-pulsed autologous DCs. UCB-NK cells were induced from CD34+ cells by stem cell factor plus IL-15. UCB-T cells were primarily activated by anti-CD3 monoclonal antibody. All those effectors were cultured with IL-2 (100 U/ml), and their cytotoxic activities were evaluated by 51Cr-release assay. UCB-NKT cells were cultured with IL-12, IL-18 or higher dose of IL-2 (1000 U/ml), and again tested for the cytotoxicity against selected targets. Results: UCB-NKT cells exhibited a pattern of killing activity against various hematological malignancies similar to that of UCB-NK cells, but could not kill K562, which was a vulnerable target for NK cells. The level of activity was quite similar to that of PB-NKT cells. In contrast, OKT-3-activated UCB-T lymphocytes showed a stronger and wider spectrum of killing compared with UCB-NK or NKT cells. IL-12, IL-18 or a higher dose of IL-2 upregulated the activity; however several targets, including fresh leukemic cells, still remained resistant. NKT cells killed auto- or allo-DCs at a level similar to that of T cells, but could not kill allo-EBCLs, which were efficiently killed by T cells. While NK cells showed only marginal or no killing against DC or EBCLs. Discussion: The anti-cancer activity of human NKT cells depends on the concentrations or the combination of Th1-cytokines. Basically, those cells might not be contributing to the immune surveillance of hematological malignancies, as shown by a relatively low cytotoxicity against malignant cells, together with the quite strong killing against auto-DCs. [ABSTRACT FROM AUTHOR]

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 (m¯eprin, A¯5 protein, receptor protein-tyrosine phosphatase μ¯) 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. [ABSTRACT FROM AUTHOR]

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-affin-ity 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 Caa+-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. [ABSTRACT FROM AUTHOR]

A 2.4-kb genomic clone, which encodes the precursor of a snake neurotoxin, erabutoxin c, was isolated from the liver of the sea snake, Laticauda semifasciata. The erabutoxin c gene is about 1.2 kb long and consists of three exons and two introns. The first intron is found at the position corresponding to the signal peptide between amino acid residues 18(Leu) and 19(Gly). The second intron is located at the position corresponding to the central loop of the mature toxin, between amino acid residues 33(Arg) and 34(Gly). A TATA box consensus sequence, characteristic of promoter regions, is identified 29–33 nucleotides upstream from the transcription-initiation site identified by nuclease S1 analysis. The erabutoxin c cDNA nucleotide sequence, deduced from the present work, is compared with the cDNA sequences of erabutoxins a and b reported previously. Replacements are found at three positions, two of which correspond to the amino acid replacements found among the toxins, while the other is silent. [ABSTRACT FROM AUTHOR]

BSTRACT The effect of ionic strength on the thermal gelation process of myosin was investigated by dynamic viscoelasticity measurements. The dynamic viscoelastic behavior of myosin was divided into three ionic strength groups. Each ionic strength group was closely related to the state of myosin molecules before the rise in temperature. Both the head and the tail portions of the molecule participated in the gel formation of myosin, but the temperature ranges differed. It was proposed that the first development of gel elasticity of myosin (30-45 °C) was attributed many to the tail portions of the molecules and that the second development (above 50 °C) was mainly to the head portions. [ABSTRACT FROM AUTHOR]

BSTRACT To elucidate the roles of the head and the tail portions of the molecule in the thermal gelation of myosin, the gelation characteristics of heavy meromyosin (HMM) and of light meromyosin (LMM) were investigated. The aggregation process of HMM corresponded to that of myosin alone in the temperature range above 50°C. Both the dynamic viscoelastic behavior and the aggregation process of LMM agreed fairly well with those of myosin alone in the temperature range up to 45 °C. Therefore, the first development of gel elasticity of myosin was attributable mainly to the tail portion of the molecule and the second was to its head portion. [ABSTRACT FROM AUTHOR]

BSTRACT The thermal gelation processes of the myosin-natural actomyosin system were investigated to determine the role of F-actin in thermal gelation of actomyosin. The dynamic viscoelastic behavior during thermal gelation changed considerably depending on the (F-actin)/ (myosin) ratio. F-actin gave the viscosity to an actomyosin sol but did not affect the elasticity development occurring in the 30 - 46°C range. The decrease in storage modulus in the 46 - 53°C range was directly induced by the presence of F-actin. The gel of myosin alone showed the highest elasticity, while that of myosin containing a small amount of F-actin had the highest elastic modulus. [ABSTRACT FROM AUTHOR]

BSTRACT Even when actomyosin dissociated into myosin and actin in the paramyosin-natural actomyosin system, the indices of gel properties increased on increasing the paramyosin content; however, their increases were smaller. Also, in the paramyosin-myosin system gels, the indices of gel properties increased on increasing the paramyosin content; however, the increases were smaller than those for the paramyosin-natural actomyosin system gels. In both systems, a high linearity was observed between each index of gel properties and paramyosin. It was likely that the paramyosin-myosin interaction did not bring about the characteristic texture of invertebrate muscle gel directly, but the binding of myosin and actin might be indispensable if the contribution of paramyosin was to occur. [ABSTRACT FROM AUTHOR]

To determine the contribution of tropomyosin to fish muscle gel characteristics, the gel properties of the tropomyosin-desensitized actomyosin system and of the tropomyosin-myosin system were investigated. For both systems, the indices of gel properties decreased considerably on increasing the tropomyosin content. Even when the two-step heating was carried out, the indices of gel properties decreased considerably on increasing the tropomyosin content. Tropomyosin negatively affected the gel formation of fish muscle and reduced the gel strength and elasticity of the fish muscle gels. [ABSTRACT FROM AUTHOR]

An expression cDNA library was constructed with poly(A)-rich RNA extracted from the collar of the giant African snail, Achatina fulica Férussac. A 1.9-kbp cDNA clone encoding a precursor of antibacterial glycoprotein of the snail, achacin, was isolated from the cDNA expression library. The cDNA sequence contains an open reading frame with 1593-nucleotide residues. The deduced amino acid sequence of this achacin precursor starts with a 29-residue leader peptide followed by a 502- residue mature peptide (56 kDa) with four possible N-glycosylation sites, Asn-Xaa-Ser or Asn-Xaa-Thr. The Northern-blot analysis proved that the achacin precursor was specifically expressed in the tissue of snail collar and processed to mature achacin, cDNA inserts encoding achacin precursor were subcloned into expression plasmids. Three kinds of expressed polypeptides were cross-reacted with rabbit antiserum raised against achacin. The largest polypeptide (Mr 63000) should be the achacin precursor. [ABSTRACT FROM AUTHOR]

A novel isoform of myosinase was purified to homogeneity from liver of spear squid by sequential chromatographies using SP Sephadex, hydroxylapatite, Zn/Co chelating affini-ty, 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-ter-minal sequence. The sequence showed similarity to an internal sequence of the astacin family. [ABSTRACT FROM AUTHOR]