Satoshi Kanoh

Thermal gelation characteristics of breast and thigh muscles of spent hen and broiler and their surimi

Spent hen (98 weeks) and broiler (12 weeks) breast and thigh muscles were minced (1 mm orifice diameter) and washed with 0.1% NaCl. A portion of both unwashed and washed mince was mixed with cryoprotectants (CP) at the rate of 4% sucrose, 4% sorbitol, and 0.2% Na-tripolyphosphate to produce surimi and kept frozen at -20°C. The mince and surimi were ground with 3% NaCl and a small amount of water to adjust the final moisture content of 80%. The pastes were stuffed into the sausage casing and heated at 90°C for 15 min to produce gel. The effects of washing, heating and CP on colour composition and thermal gelation properties of hen and broiler minces and surimi were compared. Broiler mince was lighter and less red in colour, higher in protein and lower in moisture, lipid and collagen. Gel strength and breaking strength were higher in spent hen surimi compared to broiler surimi under similar gelation conditions. Gel elasticity, springiness and water retention properties were almost identical in two surimi. Gel quality was markedly deteriorated in spent hen surimi but not so in broiler surimi after 8 weeks frozen-storage. Although CP increased the gel strength of fresh surimi (non-frozen, 0 week storage) from both hen and broiler, they were more effective in broiler surimi than hen surimi in protecting the functional quality of gel.

Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch

SUMMARY Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini, respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.

Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature

1. 1. Myosin and its subfragment-1 (Sl) from carp acclimated to 10°C showed higher actin-activated Mg2+-ATPase activity and lower thermostability than their counterparts from carp acclimated to 30°C. Accordingly, filament velocity for the 10°C-acclimated carp myosin was higher at any measuring temperatures from 3 to 23°C than that for the 30°C-acclimated carp myosin. 2. 2. Three types of cDNA clones encoding myosin heavy chains were isolated from thermally acclimated carp. The 10 and 30°C types were predominating in carp acclimated to 10 and 30°C, respectively, whereas the intermediate type was found as a minor component in the 10°C-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 30°C types. 3. 3. The three types of myosin rod all showed a typical coiled-coil structure of α-helices. DSC scans demonstrated that myosin rod prepared from carp acclimated to 10°C had a lower thermostability than that from carp acclimated to 30°C, showing that low thermostability in cold-acclimated carp myosin prevails over the entire molecule. 4. 4. cDNA clones encoding myosin alkali light chains were isolated from thermally acclimated carp. Northern blot analysis showed that the ratios of LC3LC1 mRNAs were significantly higher (3.92) in the 30°C- than 10°C-acclimated (3.10) carp.

Twitchin as a regulator of catch contraction in molluscan smooth muscle

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.

Novel Genes Participating in the Formation of Prismatic and Nacreous Layers in the Pearl Oyster as Revealed by Their Tissue Distribution and RNA Interference Knockdown

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399–4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a Ka of (2.44 ± 0.09) × 105 m−1 with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a Ka of (5.83 ± 0.05) × 104 m−1 with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca2+ sensitivity in that in the presence of Ca2+ complex formation was reduced.

Effects of urea on actin-activated Mg2+-ATPase of requiem shark myosin

Abstract The influence of urea on actin-activated myosin Mg 2+ -ATPase of requiem shark Triakis scyllia was examined and compared to that of carp ( Cyprinus carpio ). No changes in the maximum turnover rate, V max , was observed for shark myosin up to 0.3 M urea which is an approximate physiological concentration in marine elasmobranch tissues. The rate for carp myosin decreased by 70% at this urea concentration. The affinity of myosin to actin also remained unchanged up to about 0.7 M urea for shark in marked contrast to carp myosin which decreased by 30%, even in 0.1 M urea. Since it has been reported that urea-resistibility of requiem shark myosin Ca 2+ -ATPase in the absence of actin is comparable to that of carp, a high resistibility against urea in actin-activated Mg 2+ -ATPase of shark myosin is partly accounted for by its stable interaction with actin.

Genome-wide survey of genes encoding muscle proteins in the pearl oyster, Pinctada fucata.

The mechanisms of contraction of molluscan striated and smooth muscles differ from those in vertebrates. Molluscan striated muscles adopt a myosin-linked regulation, unlike vertebrates. Smooth muscles in these species show a unique form of contraction, in which the tension is maintained for a long time with little energy consumption, called catch. The available gene information is insufficient to elucidate the mechanism of contraction of molluscan muscles at the molecular level. BLAST searching was thus used to annotate genes encoding proteins related to muscle contraction in the completely determined genome of the pearl oyster Pinctada fucata using partial nucleotide sequences obtained by 3′ RACE. We identified genes that encode components of the thick-filament, such as myosin heavy chain, myosin essential and regulatory light chains, paramyosin and twitchin; of the thin-filament, such as actin, tropomyosin, troponin-T, troponin-I, troponin-C and calponin; and the PKA catalytic subunit, which is a key player in the regulation of catch contraction. The analysis indicated that isoforms of myosin heavy chain, paramyosin, and calponin are produced by alternative splicing.

Gel Forming Ability and Other Properties of Eleven Underutilized Tropical Marine Fish Species

Abstract The gel forming ability and other characteristics of the mince of 11 underutilized marine fish were studied. They were Bombay duck, silverbelly, sea catfish, silver jewfish, jewelled shad, queenfish, Spanish mackerel, hardtail, Indian tuna, tripletail and false conger eel. Mince was prepared from fillet and a portion of the mince was washed two times with cold water (5°C) containing 0.1% NaCl. Both washed and unwashed mince were ground with 3% NaCl. Ground paste was then stuffed into plastic tube and heated for one- and two-step heating. In the one-step heating, the tubes were subjected to 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. In the two-step heating, the tubes were pre-heated at 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. After the pre-heating, the tubes were immediately subjected to 85°C for 30 min. The gel was subjected to puncture, folding, expressible moisture and sensory tests. Two-step heating distinctly improved the gel strength compared to the one-step heating. The improvement due to two-step heating was more at low preheating temperatures from 25-35°C. Washing improved the texture and color of all of the gels except Bombay duck and decreased the extent of gel-disintegration in silverbelly, queenfish, sea catfish and hardtail. The gels were set optimally at 35°-40°C for most species. Species variation in the disintegration of the gels was observed. Bombay duck mince produced very weak gel. Neither two-step heating nor washing could improve the gel quality of Bombay duck mince. Our data suggested that jewelled shad, queenfish, silver jewfish, sea catfish, tripletail and false conger eel could be suitable as the material for surimi.

Possible utilization of the pearl oyster phospholipid and glycogen as a cosmetic material

Abstract The inner contents of pearl oysters from which pearls of high commercial value are harvested do not find any effective use. We screened the inner contents of pearl oyster Pinctada fucata for surfactants or humectants which may be commercially useful as a cosmetic material. Surface tension of the total phospholipid fraction and column-separated subfractions from the pearl oyster were 30-48 mN/m and comparable to those of commercially available surfactants for cosmetic use. Furthermore, the cell proliferation rate and collagen synthetic ability were increased by 11-13% and 29-61%, respectively, when crude glycogen from the pearl oyster was supplied to the culture medium for fibroblasts at concentrations of 0.01-0.2%. When β-particles prepared from the pearl oyster crude glycogen were added to the culture medium for fibroblasts at a concentration of 0.1%, the cell proliferation rate and collagen synthetic ability increased by 15% and 38%, respectively. In addition, the recovery from ultraviolet damage for epidermal keratinocytes was increased by 15-21%, when the crude glycogen from the pearl oyster was added at concentrations of 0.05-0.20% to the culture medium. These results suggest that the phospholipid and crude glycogen from pearl oyster are promising candidate as a cosmetic material.