Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

SUMMARY: A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg²⁺-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg²⁺-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg²⁺-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

Preparation of heavy meromyosin from the autolyzed squid mantle muscle homogenate

ABSTRACT:   Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg²⁺-ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca²⁺, possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca²⁺. Ca²⁺ stabilized HMM especially in a bound form to F-actin.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

Characterization of fast skeletal myosin from white croaker in comparison with that from walleye pollack

ABSTRACT:   Enzymatic and structural properties of white croaker fast skeletal muscle myosin were determined and compared with those of walleye pollack counterpart. Ca²⁺-ATPase activity of white croaker myosin was decreased to approximately 70% of the original activity during 1 day of storage at 0°C and pH 7.0 in 0.5 M KCl and 0.1 mM dithiothreitol, whereas that of walleye pollack was decreased to approximately 20% under the same condition. The activation energy ( E _a) for inactivation of white croaker myosin calculated by the Arrhenius plot for inactivation rate constant (K_D) was 1.2-fold higher than that of walleye pollack. While Ca²⁺-ATPase showed a similar KCl-dependency for the two species, the maximal activity was observed at pH 6.2 and 6.3 for white croaker and walleye pollack, respectively. Actin-activated myosin Mg²⁺-ATPase activity of white croaker was approximately half that of walleye pollack at 0.05 M KCl and pH 7.0, although the two myosins showed a similar affinity to F-actin with K _m of 1.7 and 1.4, respectively. Limited proteolysis with α-chymotrypsin cleaved heat-denatured white croaker myosin mainly at heavy meromyosin/light meromyosin (HMM/LMM) junction, whereas walleye pollack myosin was cleaved at several sites in LMM as well as at the HMM/LMM junction.     

Delay of the egg activation process in the Black Tiger Shrimp Penaeus monodon by manipulation of magnesium levels in spawning water

The aim of this study was to determine whether magnesium (Mg²⁺) in seawater is required for egg activation of the black tiger shrimp Penaeus monodon and whether manipulation of Mg²⁺ levels can be used to delay the process and thereby synchronize egg activation. Female P. monodon broodstock were allowed to spawn in artificial seawater containing Mg²⁺ at varying levels with respect to the normal (100%) level: 100%, 50%, 20% and 0%. Egg activation occurred normally at 100% Mg²⁺, incompletely at 50% and 20% Mg²⁺ levels and did not occur at all with 0% Mg²⁺. The fertilization rate with 100% Mg²⁺ was observed to be 83%, but fertilization failed to take place in all the other groups. The fertilization rate was restored from 0% to 76% following the 20% Mg²⁺ level treatment when Mg²⁺ levels returned to normal (100%) as soon as spawning was completed. This study suggests that the level of Mg²⁺ in seawater plays a vital role in P. monodon egg activation, and that commencement of this process could be delayed by manipulation of the Mg²⁺ level during and immediately after spawning.     

Immunolocalization of Na+, K+-ATPase-rich cells in the gill and urinary system of Persian sturgeon, Acipenser persicus, fry

Localization of Na⁺, K⁺-ATPase-rich cells in the gill and urinary system of Acipenser persicus fry was performed through immunofluorescence light microscopy using a mouse monoclonal antibody IgGα₅ raised against the α-subunit of chicken Na⁺, K⁺-ATPase. Different types of epithelia were clearly identified in the gill epithelium: epithelia of branchial arch, interbranchial septum, filament and lamellar epithelium. The Na⁺, K⁺-ATPase-rich cells were found in the epithelia of branchial arch, interbranchial septum, filament, interlamellar region and also in the lamellae. Histologically, the urinary system is divided into head kidney, trunk kidney and short caudal kidney. The head kidney is composed of the pronephric tubules and the haemopoietic tissues, while the trunk kidney is composed of a large number of glomeruli and convoluted nephrons. Each nephron consisted of a large glomerulus and tubules (neck, proximal, distal and collecting tubules) which connected to ureters. Posteriorly, ureters extended and joined together to form a small urinary bladder. In the urinary system, no specific fluorescence staining was observed in the glomerulus, neck segment and proximal tubules. The distal tubule cells and collecting tubule cells showed a strong immunostaining of Na⁺, K⁺-ATPase. Epithelia of ureters and urinary bladder also showed several isolated immunofluorescent cells. Immunofluorescent cells were rich in Na⁺, K⁺-ATPase enzyme which is very important for osmoregulation.     

Comparison of physiological smolt status in descending and nondescending wild brown trout (Salmo trutta) in a Danish stream

Abstract –  Downstream movement of a wild population of brown trout was examined in a small Danish stream in relation to morphological and physiological smolt status from March to May. Downstream movement was monitored in a Wolf-type trap covering all possible passage routes in the stream. Trout caught in the trap were classified as parr, pre-smolt or smolt based on morphological criteria and compared with trout randomly caught by electrofishing upstream of the trap. Representative gill samples from trap-caught and electrofished trout were analysed for gill Na⁺, K⁺-ATPase activity and used as a measure of physiological smolt status. Only a few parr occurred in the trap. Few pre-smolts occurred in the trap evenly in March and early April. In late April, pre-smolt movement peaked. By comparison, the main downstream movement of smolts occurred in distinct peaks through late March and April. The majority of fish caught in the trap were judged as pre-smolts or smolts based on morphological criteria's and they were characterised by relatively high gill Na⁺, K⁺-ATPase activity compared with trout judged as parr. Trout caught by electrofishing upstream the trap, were classified as parr, pre-smolts and smolts early in the season (March). During and after the main smolt-run in April the distribution of the remaining trout in the brook became skewed in favour of pre-smolt and parr. The study suggests that smolting trout initiate downstream movement once having reached a certain physiological smolt condition (judged by increased gill Na⁺, K⁺-ATPase activity).     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

ABSTRACT:    Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca²⁺-, Mg²⁺- and K⁺(EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca²⁺ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity.     

Influence of interposition of pink muscle fiber into dorsal ordinary muscle on 5'-IMP degrading activity in various fish species

ABSTRACT: To clarify the cause of differences in the temporal change of K -value among fish species living in the same habitat water temperature, the influence of the interposition of pink muscle fibers into dorsal ordinary muscle on 5'-inosine monophosphate (5'-IMP) degrading activity was examined. Fourteen fish species from the Sakishima Islands (habitat water temperature 28°C) and from Nagasaki (habitat water temperature 17°C) were used for the sample fishes. Each of 5'-IMP and ρ-nitrophenol phosphate (ρ-NPP) degrading activities showed a peak at near pH 8.0 and near pH 5.0, respectively. These activities were somewhat higher in fish from Nagasaki than in fish from the Sakishima Islands. The interposition percentage of pink muscle fibers into dorsal ordinary muscle correlated significantly ( P < 0.05) with the 5'-IMP degrading activity at pH 7.0. Furthermore, the activity at pH 7.0 correlated significantly ( P < 0.001) with the increasing rate of K -value at 32°C (ΔK₃₂). These results suggest that the difference in ΔK₃₂ among fish species in the same habitat water temperature might be caused by differences in 5'-IMP degrading activity because of the differences in the interposition rates of pink muscle fibers into dorsal ordinary muscle.     

Influence of interposition of pink muscle fiber into dorsal ordinary muscle on temporal change of K-value in cultured carp

To clarify the influence of the interposition of pink muscle fiber into dorsal ordinary muscle on temporal change of K-value, using cultured carp, the dorsal muscle was divided into five muscle parts towards depth with the naked eye as follows: the dark muscle part (P-1), the intermediate muscle part (P-2) and three ordinary muscle parts (P-3, P-4, P-5). These were organized from the muscle fiber types as follows: P-1 was only red muscle fiber type. P-2 was only pink muscle fiber type in a thin layer and two muscle fiber types of not only pink muscle fiber but also white muscle fiber of the IIa or IIb subtype in a mosaic pattern. All of P-3, P-4 and P-5 were two muscle fiber types, white muscle fiber (IIa or IIb subtype) and pink muscle fiber. The temporal changes of K-values were remarkably faster in the order of P-1, P-2, and three parts of P-3, P-4 and P-5. The changes did not exhibit a remarkable difference among the three ordinary muscle parts. From these results, it was considered that the interposition of pink muscle fiber into the dorsal ordinary muscle might accelerate the temporal change of K-value.     

Effects of Temperature on Growth and Protein Assimilation in Juvenile Leader Prawns Penaeus monodon

Abstract.— The effects of temperature on growth and protein assimilation in leader prawns Penaeus monodon were evaluated. Growth of leader prawns was significantly ( P < 0.05) affected by temperature. The highest growth rate was 0.124 g/d at 32.0 C and optimum growth occurred between 27.5–32.0 C. Growth was described by an equation of the form: W _t = W_oe^kt ( R² = 0.99). The relationship between temperature and growth was described by a curvilinear equation of the form: Y = a + bX + cX² ( R² = 0.70). Apparent protein assimilation efficiency was not significantly ( P > 0.05) affected by temperature.     

Effects of the probiotic, Lactobacillus acidophilus, on the growth performance, haematology parameters and immunoglobulin concentration in African Catfish (Clarias gariepinus, Burchell 1822) fingerling

This experiment was carried out to evaluate the effects of the probiotic, Lactobacillus acidophilus , on the growth performance, haematology parameters and immunoglobulin concentration in African catfish Clarias gariepinus fingerling. Two experimental diets were formulated to contain 35 g kg⁻¹ crude protein and 10 g kg⁻¹ lipids accordingly and fed three times daily for 12 weeks to 25 C. gariepinus fingerlings per fibreglass tank in 12 replicates each. The control diet was prepared with no probiotic supplementation whereas the second diet was prepared supplemented with a probiotic, L. acidophilus , containing about 3.01 × 10⁷ colonies/g of diet. The results show that growth performance [specific growth rate (SGR) and relative growth rate (RGR)], nutrient utilization [protein efficiency ratio (PER) and feed conversion ratio (FCR)] and survival were significantly ( P <0.05) higher in fish maintained on the probiotic-supplemented diet compared with those on the control diet. Haematology parameters (packed cell volume, haemoglobin, erythrocyte sedimentation rate, red blood cell and white blood cell, total serum protein, Ca²⁺, Mg²⁺, Cl⁻, glucose and cholesterol) and total immunoglobulin concentrations were also significantly better in fish fed the probiotic-supplemented diet than in the control. Although the water quality parameters monitored were better in the fish fed the probiotic-supplemented diet than in the control, the parameters were not significantly different ( P >0.05). From the results of this experiment, we conclude that L. acidophilus can be used as a probiotic agent in African catfish culture, to enhance fish health, survival and better feed efficiency and growth performance.     

Potential benefit of clove oil sedation on animal welfare during salmon smolt, Salmo salar L. transport and transfer to sea

The purpose of this study was to determine the effect of clove oil (4.0 mg L⁻¹) sedation, compared with non-sedation, on the primary (plasma cortisol), secondary (osmoregulation) and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. Clove oil sedation during on- and off-loading sufficiently reduced the primary stress response to lower mortality (2.1%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 12.2%. The unsedated fish experienced an acute mortality that stabilized only 6 days after the transport. None of the secondary stress responses measured in this experiment could contribute towards explaining this phenomenon, with the possible exception of plasma magnesium (Mg²⁺). Plasma Mg²⁺ differed between the groups; while plasma Mg²⁺ in the clove oil sedated group returned to pre-stress levels 72 h after transport, the unsedated group showed no such recovery even 1 week after transport, which may indicate a disturbance in the hydromineral balance, and provides a plausible explanation for the delayed mortality in this group. Eugenol-based anaesthetics appear to be promising as a stress-reducing sedative for Atlantic salmon smolts, and, if used properly, this chemical could improve animal welfare and survivability during and after common aquaculture-related incidents.     

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Angling pressure, yield and catch per effort of grayling, Thymallus thymallus (L)., and brown trout, Salmo trutta L., on the rivers Glomma and Rena, southeastern Norway

Abstract Postal questionnaires, interviews and accurate reports from anglers were used to study angling activity, yield and catch per effort of grayling, Thymallus thymallus (L.), and brown trout, Salmo trutta L., on three stretches of the regulated river Glomma and two stretches of its tributary, the river Rena in southeastern Norway. Angling activity was expressed as hours angling per kilometre of river and varied from 364 (CL 245–598) to 1337 (CL 912–1991) h km^-1. Grayling and brown trout dominated the catches. The catches were estimated between 476 (CL 334–659) and 753 (CL 517–1155) grayling and brown trout per kilometre river stretch. The mean weights were estimated between 216 and 353 g for grayling, and 260 and 395 g for brown trout. The yield of grayling and brown trout was estimated between 8.7 (CL 6.1–12.4) and 46.4 (CL 34.9–62.3) kg ha^-1 yr^-1. The quotient grayling/brown trout in the catches varied between 0.41 and 5.1, and was found to be significantly negatively correlated with angling effort ( r = - 0.838, P <0.05). Catch per effort was estimated between 132 (CL 82–218) and 382 (CL 287–513) g h^-1, and was also negatively correlated to angling effort, according to: Catch per effort (g h^-1) = - 0.210 × effort (h km^-1) + 405 ( r = and 0.899, P <0.02).
Angling activity in earlier years was determined from number of permits sold where data were available. On stretches where angling activity was moderate, there was a tendency towards increase, whereas on stretches where activity had been high for several years, the activity seemed to have stagnated, indicating that a saturation had been reached.     

Cage culture of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) at different stocking densities in a shallow eutrophic lake

Postlarvae of Litopenaeus vannamei were acclimated and stocked in lake-based cages at the following stocking densities: 10, 20, 30 and 40 shrimp m⁻². Another set of shrimp was stocked in concrete tanks as reference samples at 30 shrimp m⁻². Significant differences were observed among stocking densities throughout the 95-day culture. The final weight at harvest decreased with increasing stocking density: mean weights of 23.3, 15.8, 13.0, 10.9 and 14.6 g for the 10, 20, 30, 40 shrimp m⁻² and reference tanks were observed respectively. There were no significant differences in survival throughout the culture period, ranging between 69% and 77%. Daily growth rates (range: 0.11–0.24 g day⁻¹) and specific growth rates (range: 3.54–4.34%) also differed significantly among stocking densities, both increasing with decreasing stocking density. The feed conversion ratio in the cages did not differ among the stocking densities, ranging from 1.53 to 1.65. The relationship between stocking density and mean individual weight at harvest followed the equation y =81.06 x ^−0.54 ( R ²=0.938) and that of stocking density and production (in g m⁻²) is y =58.01 x ^−0.46 ( R ²=0.834).     

Urine properties in carp, Cyprinus carpio L., with sekoke disease

Abstract. Changes in the properties of urine and blood of carp with sekoke disease were studied. With the progress of this disease, the parameters of blood and particularly urine changed significantly. After 120 days, urine flow, osmolarity and concentrations of inorganic ions (K⁺, Ca²⁺, Mg²⁺, Cl⁻) and organic compounds (ammonium, creatine and creatinine, protein, glucose) in the experimental group showed higher values than those of the controls. Even after 60 days, when clinical signs of sekoke disease were not evident, abnormally high concentrations of ammonium, protein and glucose in the urine were found in the experimental group.     

Effects of lipid extraction on stable carbon and nitrogen isotope analyses of fish tissues: potential consequences for food web studies

Abstract –  We examined whether solvent-based lipid extractions, commonly used for stable isotope analysis (SIA) of biota, alters δ ¹⁵N or δ ¹³C values of fish muscle tissue or whole juvenile fish. Lipid extraction from muscle tissue led to only small (<1‰) isotope shifts in δ ¹³C and δ ¹⁵N values. By contrast, ecologically significant shifts (+3.4‰ for δ ¹³C and +2.8‰ for δ ¹⁵N) were observed for whole juvenile fish. Sample variance was not affected by lipid extraction. For tissue-specific SIA, two sample aliquots may be required: a lipid-extracted aliquot for stable carbon isotope analysis when differing lipid content among tissues is a concern, and a nonextracted aliquot for δ ¹⁵N determination. Whole organism SIA is not recommended because of the mix of tissues having different turnover times; for very small fish, we recommend that fish be eviscerated, decapitated, and skinned to minimise differences with samples of muscle tissue.