Postmortem Degradation of White Fish Skeletal Muscle (Sea Bass, Dicentrarchus labrax): Fat Diet Effects on In Situ Dystrophin Proteolysis During the Prerigor Stage

All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4°C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0°C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations. Received May 4, 2000; accepted October 15, 2000     

Production and Characterization of a Continuous Embryonic Cell Line from Sea Bass (Dicentrarchus labrax L.)

Continuous cell lines represent an important tool both for biological studies and for their applications in marine biotechnology. In this article we describe the production and characterization of a continuous adherent cell line, named DLEC, derived from early embryos of the European sea bass Dicentrarchus labrax L. (Actinopterygii, Moronidae). Cells were obtained by disrupting 2- to 12-hour-old embryos and culturing resulting cells at 18°C in RPMI medium containing 5% fetal calf serum (FCS) and 10% supernatant fraction of the embryo homogenate. After 8 weeks culture medium was replaced with Liebovitz's L15 medium containing 10% FCS and DLEC cells started proliferation. Subsequently, they were continuously cultured until the 50th passage without evident changes in their morphology. DLEC cells show a fibroblast-like shape and a modal chromosome number of 48, as do the wild-type cells; conversely the constant presence of six to nine meta-submetacentric elements in the karyotype (vs. zero to two in the wild-type) indicates the occurrence of chromosomal rearrangements during stabilization. DLEC cells are sensitive to substances known to induce differentiation of mammalian cells such as retinoic acid and phorbol esters. They have been transfected using liposomes with a commercial plasmid vector containing a reporter gene, thus suggesting a possible importance as an alternative expression system of recombinant vertebrate proteins in teleost cells.     

Variation in Gillraker Number During Growth of the Sea Bass, Dicentrarchus labrax (Perciformes: Moronidae), Reared at Different Salinities

A marked salinity-related decrease in gillraker number in sea bass samples during growth was documented. All specimens share the same genetic stock and rearing conditions were constant during early ontogeny. Variation in gillraker number could be related to the ecophenotypism of this character but selection can not be excluded. Results obtained from reared specimens were compared with those reported in the literature and with data collected from wild stocks. Attention is focused on the pitfalls that the use of this character may have on the taxonomy of fish species.     

The Acclimation of European Sea Bass (Dicentrarchus labrax) to Temperature: Behavioural and Neurochemical Responses

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28°C). After 21 d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28°C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28°C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22°C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28°C in comparison with those maintained at 18°C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28°C than at 18°C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.     

Antioxidant Peptides from the Protease Digest of Prawn (Penaeus japonicus) Muscle

The muscle of the prawn Penaeus japonicus was hydrolyzed by various proteases, and antioxidant activity of the hydrolysates was examined. Among the digests, pepsin digest showed the most potent antioxidant activity. Three antioxidant peptides have been isolated from the active peptidic fraction by ion-exchange chromatography, gel filtration, and ODS high-performance liquid chromatography. Their structures were identified as Ile-Lys-Lys, Phe-Lys-Lys, and Phe-Ile-Lys-Lys. Received October 16, 1998; Accepted January 8, 1999.     

Light and Electron Microscopic Description of Sphaerospora dicentrarchi N. Sp. (Myxosporea: Sphaerosporidae) from Wild and Cultured Sea Bass, Dicentrarchus labrax L.

ABSTRACT. A new myxosporean, Sphaerospora dicentrarchi n. sp., was found in numerous organs of wild and cultured sea bass ( Dicentrarchus labrax L.). It is distinguished from all previously reported Sphaerospora spp. by the shape and small size of the spores, location in the host and its geographical distribution. Prevalence of infection was 100% and 83.5% in wild and cultured fish, respectively. Outstanding ultrastructural features are the presence of a binucleate sporoplasm, valvogenic spheroidal structures and capsulogenic lipid inclusions. Other data concerning ultrastructure and sporogenesis are presented.     

鲈血清免疫球蛋白分离纯化及部分特性分析

采用硫酸铵盐析、透析和Sephadex G-200凝胶过滤的方法纯化出鲈(Lateolabrax japonicus)血清免疫球蛋白,利用高效液相色谱(HPLC)结合SDS-PAGE的方法分析其性质。HPLC结果表明：纯化得到的IgM在经巯基乙醇处理后解离形成轻链和重链两个亚单位,SDS-PAGE确定其分子量分别为72ku和28ku。实验说明,HPLC的检测方法具有广阔的应用前景;而对鲈IgM的分析为鱼类免疫球蛋白的比较研究提供了基础资料。     

Purification and Characterization of the Lectin from Taro (Colocasia esculenta) and Its Effect on Mouse Splenocyte Proliferation In Vitro and In Vivo

Lectins are proteins found in a wide range of organisms, with the ability to bind reversibly to specific carbohydrates. They can display important biological activities, such as the activation of the cell cycle in lymphocytes. Storage proteins with lectin activity have been reported in tuberous plant species, such as Colocasia esculenta, popularly known as taro. A simple strategy based on Cibacron Blue chromatography was used to purify a 12 kDa polypeptide 1.3-fold, with a recovery of 30 %. The purified protein was identified as tarin by mass spectrometry, which indicated that it was present in G1a/G1d isoforms. Tarin exhibited both agglutinating activity against hamster erythrocytes and mitogenic activity in vitro and in vivo toward mouse splenocytes. Optimum cellular proliferation in vitro was achieved by 625 ng of the crude extract or 500 ng of the purified tarin. Total mouse splenocyte proliferation measured after 5 days of intraperitoneal inoculation of purified tarin was increased 3.3-fold in comparison to the control group. Half of the proliferating cells were identified as B lymphocytes by flow cytometry. These results show that this is an efficient and simple strategy to purify tarin and aid in establishing this protein as a new therapeutic drug, able to promote cell proliferation in a murine model.     

Purification,Characterization, and Antiproliferative Activity of a Single-Chain Lectin from Vicia palaestina (Fabaceae) Seeds

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity.     

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.     

Expression and Characterization of European Sea Bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) Somatolactin: Assessment of In Vivo Metabolic Effects

Abstract The complementary DNA coding for European sea bass somatolactin was expressed in the pET-3a bacteria expression vector. The recombinant somatolactin (rbSL) was purified by size exclusion chromatography, and 95% of the protein remained in the oxidized form with negligible aggregation over prolonged cold storage. The identity of the recombinant protein was demonstrated by Western blotting with a rabbit polyclonal antibody against gilthead sea bream somatolactin. The same antibody was utilized in a radioimmunoassay procedure, using rbSL as standard and radioiodinated tracer. Curve displacements of pituitary and plasma samples paralleled the rbSL standard, and the midrange of the assay (8 ng/ml) was low enough to measure in a consistent manner the circulating SL concentration. To assess biological activity a single dose of rbSL (0.1 μg/g of body mass) was administered to juvenile gilthead sea bream by intraperitioneal injection. In comparison with saline-treated fish, rbSL did not modify the circulating amount of insulin-like growth factor I, whereas a 50% increase was found with the same dose of recombinant trout growth hormone (rtGH). Hormone treatment did not modify nitrogen-ammonia excretion, but both rbSL and rtGH increased carbon dioxide output and oxygen uptake, which in turn decreased the respiratory quotient (CO₂ output per O₂ uptake). This pattern of gas exchange suggests the enhancement of lipid catabolism, which is consistent with the observation that both hormones were able to inhibit the hepatic activity of acetyl–coenzyme A carboxylase. These new insights provide direct evidence for the involvement of fish somatolactin in energy homeostasis, which may serve to maintain the lipolytic tonus in different physiologic states.     

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP,and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.     

Real-time polymerase chain reaction, in situ hybridization and immunohistochemical localization of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes)

The distribution of insulin-like growth factor-I (IGF-I) and myostatin (MSTN) was investigated in sea bass (Dicentrarchus labrax) by real-time polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry. Real-time PCR indicated that IGF-I mRNA increased from the second day post-hatching and that this trend became significant from day 4. ISH confirmed a strong IGF-I mRNA expression from the first week post-hatching, with the most abundant expression being detected in the liver of larvae and adults. Real-time PCR also showed that the level of MSTN mRNA increased significantly from day 25. The expression of MSTN mRNA was higher in muscle and almost absent in other anatomical regions in both larvae and adults. Interestingly, the lateral muscle showed a quantitative differential expression of IGF-I and MSTN mRNAs in red and white muscle, depending on the developmental stage examined. IGF-I immunoreactivity was detected in developing intestine at hatching and in skeletal muscle, skin and yolk sac. MSTN immunostaining was evident in several tissues and organs in both larvae and adults. Both IGF-I and MSTN proteins were detected in the liver from day 4 post-hatching and, subsequently, in the kidney and heart muscle from day 10. Our results suggest, on the basis of a combined methodological approach, that IGF-I and MSTN are involved in the regulation of somatic growth in the sea bass. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This research was supported by grants from the Italian Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MIUR) and by the University of Padua (Progetto di Ateneo).     

Purification and Characterization of Mannitol-l-Phosphatase in the Red Alga Caloglossa continua (Ceramiales, Rhodophyta)

Purification of mannitol-l-phosphatase, an enzyme catalyzing the final step of mannitol biosynthesis, was first achieved in the mannitol-accumulating red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was shown to be a monomer, since gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave close values of apparent molecular weights of 28,500 and 30,200, respectively. The protein exhibited an isoelectric point of 4.8. The substrate specificity for mannitol-l-phosphate (MIP) was very high, and that for K _m(MIP) was 0.41 mM. The catalytic activity was optimal at pH 7.4. The enzyme was activated by Mg²⁺, but was strongly inhibited by Ca²⁺, NaF, N-ethylmaleimide, and p-hydroxymercuribenzoic acid. Seawater levels of NaCl and physiological levels of mannitol also inhibited the activity by 50% or more. Changes in the concentrations of those ions and metabolites may regulate the biosynthesis of mannitol as an osmoregulant in vivo. Received May 7, 2001; accepted June 15, 2001.     

Trace elements in the muscle,ova and seminal fluid of key clupeid representatives from the Gdansk Bay (South Baltic Sea) and Iberian Peninsula (North-East Atlantic)

BackgroundBaltic herring and European sardine are pelagic, fish of particular ecological importance, on the one hand control numbers of planktonic organisms, and on the other hand exist as food for predators on higher trophic levels. Moreover, these fish are among the main species caught for human consumption. Rare earth elements (REEs) come mainly from geogenic sources but, due to their use in technology, agriculture and medicine, the importance of anthropogenic sources is growing steadily.MethodsSamples used for the study were available on the market. Fresh materials of fish muscle, ova and seminal fluid were mineralized and elements were determined by means of inductively coupled plasma – mass spectrometry (ICP-MS).ResultsThe conducted research indicated the presence of REEs in the muscles of the Baltic herring (∑REE = 0.076 ± 0.047 mg/kg) and European sardine (∑REE = 0.191 ± 0.163 mg/kg), with a clear dominance of heavy REEs in both fish species. Trace elements (TE) in the muscles of the tested fish demonstrated a similar system of concentration (Baltic herring: Zn > As > Se > Cu > Cr > Ni > Pb > Cd; European sardine: Zn > As > Se > Cu > Ni > Cr > Pb > Cd). REEs and TEs in these fish were presence in ova and seminal fluid indicates intergenerational transfer.ConclusionChanges in the concentrations of some trace elements (As, Cu, Cd) in the muscles of herring indicate increases compared to the historical data. The availability of metals in the aquatic environment may be determined by ongoing climate changes, effected water salinity and warming increased availability of labile forms of trace metals. Decline trends in the condition of pelagic fish need to extend the research in the context of contemporary environmental threats.     

The fisheries biology of flying fishes (Families: Exocoetidae and Hemiramphidae) from the Camotes Sea, Central Philippines

The catches of a small artisanal fishery for flying fishes (Families Exocoetidae and Hemiramphidae) on the Danajon Bank in the Camotes Sea. Central Visayas, were recorded during a 14-month period between 1987–1988. Catches were made using floating drive-in-nets deployed from small motorized canoes. Three species, Cheilopogon nigricans, Cypselurus opisthopus and Oxyporhamphus convexus , formed about 90% of landings. Growth, mortality and related parameters for the three dominant species in the catch were estimated from length-frequency data. Seasonal variations in catch rate and recruitment are described and thought to be linked to the two monsoon periods in the Philippines. Total mortality rates were very high and, while these may be the result of migratory movements rather than attrition, they are a cause for concern in such a highly selective fishery.     

Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy

A development-specific and elicitor-inducible acyltransferase [hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] that catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphenethylamines was purified 988-fold to apparent homogeneity from opium poppy (Papaver somniferum L.) cell-suspension cultures. The purification procedure, which resulted in a 6.8% yield, involved hydrophobic interaction and anion-exchange chromatography, followed by affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent. Purified THT had an isoelectric point of 5.2, a native molecular mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electrophoresis. The purified enzyme was able to synthesize a variety of amides due to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines. The best substrates were feruloyl-CoA (VK _m ⁻¹13.4 mkat g⁻¹ M⁻¹) and tyramine (VK _m ⁻¹6.57 mkat g⁻¹ M⁻¹). The THT activity increased during development of opium poppy seedlings, occurred at high levels in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor. Immunoblot analysis using THT mouse polyclonal antibodies did not always show a correlation between THT polypeptide and enzyme activity levels. For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide was detected. Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, root periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and anther filaments. Received: 19 January 1999 / Accepted: 3 March 1999     

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from A?r? Bal?k Lake trout gill (fCA) by affinity chromatography on a sepharose 4B‐tyrosine‐sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg‐protein)^–1, and a yield of 79.3 by using sepharose‐4B‐l tyrosine‐sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was found to be about 29.9 kDa. The kinetic parameters, K_M and V_max were determined for the 4‐nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The K_i constants for mafenide ( 1 ), p‐toluenesulfonamide ( 2 ), 2‐bromo‐benzene sulfonamide ( 3 ), 4‐chlorobenzene sulfonamide ( 4 ), 4‐amino‐6‐chloro‐1–3 benzenedisulfonamide ( 5 ), sulfamethazine ( 6 ), sulfaguanidine ( 7 ), sulfadiazine ( 8 ), and acetozazolamide ( 9 ) were in the range of 7.5–108.75 μM.