Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.

Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (ß/α)8-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

Effects of Motor-Cognitive Dual-Task Standing Balance Exergaming Training on Healthy Older Adults' Standing Balance and Walking Performance.

Objective: This study examined the effects of motor-cognitive dual-task exergaming standing balance training on healthy older adults' static, dynamic, and walking balance. Methods: Twenty-four adults older than 70 years (control group: n = 9, males = 6, balance training group: n = 15, males = 8) completed the experiment. Dual-tasking standing balance training comprised the accurate control of a ping-pong ball on a tray held with both hands, while standing on one leg (analog training) and three modules of Wii Fit™ exergaming (digital training). The duration of balance training was ∼15 minutes per day, 2 days per week for 8 weeks, in total 16 sessions. We measured one-leg standing time, functional reach distance, walking balance evaluated by the distance walked on a narrow beam (4-cm long, 4-cm wide, and 2-cm high) with single and dual tasking, habitual and maximal walking speed, and muscle strength of the hip extensor, hip abductor, hip adductor, knee extensor, and plantarflexor muscle groups in the right leg at baseline and after 8 weeks. Results: Control group decreased, but balance training group increased one-leg standing time. Only the balance training group improved functional reach distance and hip and knee extensor strength. There was no change in walking speed and walking balance in either group. In the balance training group, changes in maximal speed correlated with changes in dual-tasking walking balance and changes in one-leg standing time correlated with changes in single-tasking walking balance. Conclusion: These results suggest that 16 sessions of motor-cognitive dual-task standing exergaming balance training substantially improved healthy older adults' static and dynamic balance and leg muscle strength but failed to improve walking speed and walking balance. Balance exercises specific to walking balance need to be included in balance training to improve walking balance.

Titin-isoform dependence of titin-actin interaction and its regulation by S100A1/Ca2+ in skinned myocardium.

Titin, also known as connectin, is a large filamentous protein that greatly contributes to passive myocardial stiffness. In vitro evidence suggests that one of titin's spring elements, the PEVK, interacts with actin and that this adds a viscous component to passive stiffness. Differential splicing of titin gives rise to the stiff N2B and more compliant N2BA isoforms. Here we studied the titin-isoform dependence of titin-actin interaction and studied the bovine left atrium (BLA) that expresses mainly N2BA titin, and the bovine left ventricle (BLV) that expresses a mixture of both N2B and N2BA isoforms. For comparison we also studied mouse left ventricular (MLV) myocardium which expresses predominately N2B titin. Using the actin-severing protein gelsolin, we obtained evidence that titin-actin interaction contributes significantly to passive myocardial stiffness in all tissue types, but most in MLV, least in BLA, and an intermediate level in BLV. We also studied whether titin-actin interaction is regulated by S100A1/calcium and found that calcium alone or S100A1 alone did not alter passive stiffness, but that combined they significantly lowered stiffness. We propose that titin-actin interaction is a "viscous break" that is on during diastole and off during systole.

cDNA cloning and characterization of temperature-acclimation-associated light meromyosins from grass carp fast skeletal muscle.

The three types of cDNA clones, previously defined as the 10 degrees C, intermediate and 30 degrees C-types [Tao, Y., Kobayashi, M., Liang, C.S., Okamoto, T., Watabe, S., 2004. Temperature-dependent expression patterns of grass carp fast skeletal myosin heavy chain genes. Comp. Biochem. Physiol. B 139, 649-656], were determined for their 5'-regions which encoded at least the C-terminal half of myosin rod, light meromyosin (LMM), in fast skeletal muscles of grass carp Ctenopharyngodon idella. The deduced amino acid sequence identity was 91.1% between the 10 degrees C and 30 degrees C-types and 91.4% between the 10 degrees C and intermediate-types, whereas a high sequence identity of 97.8% was found between the intermediate and 30 degrees C-types. These three grass carp LMMs all had a characteristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)(n), where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by charged residues. However, the ratios of hydrophobic residues to the total were higher for the intermediate- and 30 degrees C- than 10 degrees C-type LMM, suggesting that the former both types may form more stable coiled-coils of alpha-helices than the latter type. These differences in the primary structures of LMM isoforms might be partially implicated in differences in the thermostabilities and gel-forming profiles of myosins from grass carp in different seasons reported previously [Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2005. Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization. Fish. Sci. 71, 195-204; Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2007. Changes in rheological properties of grass carp fast skeletal myosin induced by seasonal acclimatization. Fish. Sci. 73, 189-196].

Effect of pH and heating conditions on the properties of Alaska pollock (Theragra chalcogramma) surimi gel fortified with fish oil.

To improve the quality and functionality of emulsified surimi gels, the effect of pH and heating conditions on the properties of surimi gel fortified with fish oil was investigated. Results showed that pH conditions influenced the solubility and emulsifying properties of surimi proteins and that the gel properties were associated with the protein properties. Under direct heating, the highest gel strength was achieved at pH 8.0, in which condition the solubility was significantly higher than others. Higher emulsifying stability resulted in enhanced gel strength relative to that of the control group. However, the changes in the gel strength were not consistent under two-step heating. In addition, the expressible moisture and oil content were found to vary depending on the pH values under both heating conditions. The corresponding changes in expressible moisture and oil content could be attributed to the high protein solubility and emulsifying properties of surimi proteins. Analysis of the dynamic rheological properties of the resulting surimi paste revealed that the gelation properties varied depending on the pH conditions during the heating process. In addition, the temperatures of myosin cross-linking changed according to the structure of surimi proteins, which in turn varied depending on the pH conditions. PRACTICAL APPLICATIONS: To improve the functionality of surimi-based product, the fish oil was added to prepare surimi gel. pH and heating conditions play important roles in the gelation of fish proteins. Therefore, this study investigated the effect of combined pH and heating condition on the property of surimi gel fortified with fish oil. The emulsified surimi gel with fine texture was obtained at pH of 8-8.5; moreover, heating conditions (direct heating and two-step heating) also influenced texture of emulsified surimi gel. These results provide the evidence to produce the emulsified surimi-based product with the high gel strength, water- and oil-holding capacity.

Effect of emulsifying stability of myofibrillar protein on the gel properties of emulsified surimi gel.

Several kinds of emulsified surimi gels were prepared from different quality levels of Alaska Pollack surimi, and the relationship between the emulsifying stability (ES) of myofibrillar protein and the properties of the emulsified surimi gels was investigated. Fish oil emulsified into surimi gels enhanced the breaking strength, but this was decreased by denaturation of the surimi protein, and the rate of enhanced gel-forming ability with emulsification decreased with decreasing ES. Expressible drip also decreased with emulsification; however, increasing amounts of lipid in the expressible drip were separated out from the gel upon protein denaturation of the source surimi. Scanning electron microscopy revealed that the shape of fish oil particles became irregular and some voids caused by oil leakage were observed with increasing storage period of source surimi. The results suggested that improvement in gel properties of the emulsified surimi gels was correlated with ES as well as the level of protein denaturation.

Species composition of the genus Scombrops (Teleostei, Scombropidae) in the waters around the Japanese Archipelago: detection of a cryptic species.

Current literature states that family Scombropidae consists of a single genus Scombrops comprising three species worldwide, with two of them, Scombrops boops and Scombrops gilberti, distributed in the waters around the Japanese Archipelago. Although these two scombropids are commercially important species, little is known about the ecology of these fishes. It is difficult to discriminate between these two species based on external characteristics because of their morphological similarity. Here, we report two different morphotypes characterized by the relative growth between the otolith size and the standard length (SL) of the scombropid specimens caught in southern waters off Kyushu Island, Japan, and show the genetic relationship between the morphotypes by means of phylogenetic analyses using complete DNA sequences of the cytochrome b gene. The relationship between otolith weight and SL was significantly different between specimens < 505 mm SL and those > 550 mm SL. Phylogenetic analysis demonstrated that the sequences from these scombropid specimens formed three clades: two corresponded to S. boops and S. gilberti, while the third did not correspond to any sequence recorded in databases, suggesting that these specimens are undescribed scombropid species. Almost all the specimens with SL < 505 mm (n = 76) were identified as S. boops, and only nine as S. gilberti. On the other hand, almost all the specimens with SL > 550 mm (n = 41) fell in the unidentified group except for four specimens, whose sequences were identical to that of S. boops.

Rheological properties of fast skeletal myosin rod and light meromyosin from walleye pollack and white croaker: contribution of myosin fragments to thermal gel formation.

Myosin rod and light meromyosin (LMM) of walleye pollack and white croaker were examined for their rheological properties by measuring dynamic viscoelastic parameters. Rods from walleye pollack and white croaker increased their storage moduli (G') in the ranges of 29-43 degrees C and 31-38 degrees C, respectively, in temperature sweep analysis. Walleye pollack LMM showed no peak of G' upon heating, whereas the white croaker counterpart exhibited a single sharp peak of G' at 35 degrees C. Loss modulus (G") showed similar temperature-dependent changes for the two fish species as the case of G', irrespective of rod and LMM, although G" values were lower than those of G'. Thus, rheological properties of rod and LMM were different between walleye pollack and white croaker. Taken together with data previously reported for myosin, it was considered that both myosin rods from walleye pollack and white croaker are attributed to thermal gel formation of myosin in a low-temperature range, though in a species-specific manner.

Differences in polymer formation through disulfide bonding of recombinant light meromyosin between white croaker and walleye pollack and their possible relation to species specific differences in thermal unfolding.

Fast skeletal light meromyosins (LMMs) of white croaker and walleye pollack were prepared in our expression system using Escherichia coli and determined for their polymer-forming ability and thermodynamic properties by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and differential scanning calorimetry (DSC), respectively. White croaker LMM formed dimer by heating at 80 degrees C and showed only a single peak at 32.1 degrees C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8, and 43.9 degrees C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM where Cys491 was substituted by alanine changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4, and 43.9 degrees C. However, this point-mutated LMM formed no dimer upon heating as in the case of native LMM. These results suggest that cysteine residue(s) participates in thermal gel formation of LMM when it locates in a suitable position of the sequence.

Myosin heavy chain genes expressed in juvenile and adult silver carp Hypopthalmichthys molitrix: novel fast-type myosin heavy chain genes of silver carp.

Silver carp Hypopthalmichthys molitrix is eurythermal temperate fish, whose muscle is considered to express several types of myosin heavy chain (MYH) genes at different stages of its growth and to adjust to the environmental temperature. In this study, MYH genes expressed in the muscles of juvenile and adult silver carp were investigated. Five types of MYH cDNA clone were isolated from silver carp (H. molitrix) by RACE strategy using a set of fast-type MYH specific primers, and termed scMYH(F1), scMYH(F2), scMYH(F3), scMYH(F4) and scMYH(F5) in the order of their abundance in cDNA libraries constructed from fast skeletal muscles of adult silver carp. scMYH(F1), scMYH(F3) and scMYH(F5) showed high nucleotide sequence identities of 96, 98 and 96% to gcMYH(F30), gcMYH(F10) and gcMYH(FI), respectively, that encode MYHs predominantly expressed in fast skeletal muscle of grass carp (Ctenopharyngodon idella) acclimated to 30, 10 and 20 degrees C, respectively. scMYH(F2) and scMYH(F4) showed a high identity to A4-type MYH from rock cod (Notothenia coriiceps) slow skeletal muscle. Phylogenetic analysis demonstrated that scMYH(F1) and scMYH(F5) were monophyletic with fish adult fast-type MYHs, whereas scMYH(F2) and scMYH(F4) formed a cluster with fish slow-like fast-type MYH, and scMYH(F3) did with fish embryonic fast-type MYHs. Interestingly, juvenile silver carp predominantly expressed scMYH(F3) irrespective of acclimation temperatures at 10, 18 or 26 degrees C. The comparison among scMYH(F1), scMYH(F2) and scMYH(F3) in the deduced amino acid sequence revealed that the putative binding sites for ATP, actin, and essential and regulatory light chains in myosin subfragment-1 (S1) have high identities with each other (81-100%). However, their loop-1 and loop-2 regions in S1 were highly variable, suggesting their different functions. The deduced amino acid sequences of myosin subfragment-2 and L-meromyosin showed high identities of 90-91% and 86-90%, respectively, among the above three scMYHs.

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle.

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5'-regions of the three known genes encoding light meromyosin isoforms (10 degrees C, intermediate and 30 degrees C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 degrees C and intermediate types, between the 10 degrees C and 30 degrees C types, and between the intermediate and 30 degrees C types, respectively. Isoform-specific differences were clearly observed between the 10 degrees C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 degrees C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 degrees C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.