Biological activities of synthetic grammistins and analogous peptides

Grammistins, peptide toxins isolated from the skin secretions of two species of soapfishes Grammistes sexlineatus and Pogonoperca punctata, are new members of the family of linear membrane-lytic antibacterial peptides characterized by the abundance of amphiphilic α-helices. As the first step toward future use of grammistins to examine the mode of membrane lysis and design new antibiotics, C-terminally free and amidated forms were chemically synthesized for each of five grammistins (Gs 1, Gs 2, Pp 1, Pp 2b and Pp 3), and evaluated for hemolytic and antibacterial activities. No difference in biological activities was observed between C-terminally free and amidated forms of Gs 1, while the C-terminally amidated forms of the other grammistins exhibited much higher activities than the C-terminally free forms. Comparison with the biological activities of natural grammistins, together with our preliminary cloning experiments, showed that natural grammistins, except for Gs 1, are C-terminally amidated. As for Pp 1, six analogous peptides were further synthesized. Determination of their biological activities revealed that the amphiphilic α-helical structure and positive charge are important for the hemolytic and antibacterial activities, respectively, of Pp 1.     

Growth and biochemical composition of juvenile green abalone Haliotis fulgens fed rehydrated macroalgae

Under controlled laboratory conditions, juvenile green abalone Haliotis fulgens were fed rehydrated natural feeds to determine the effects on growth, survival, feed efficiency and biochemical composition of the digestive gland and muscle. Five macroalgae, Ulva sp. (Chlorophyta), Eisenia arborea, Macrocystis pyrifera, Egregia menziesii (Phaeophyta) and Porphyra perforata (Rhodophyta) were tested. The macroalgae promoted growth, although, depending on the species, there were considerable differences in growth, feed efficiency and biochemical composition of the digestive gland and muscle. Mean growth rate in length and weight and survival rate varied within the ranges 1.6–15.1 μm day^?1, 1.4–8.1 mg day^?1 and 44–69%, respectively. Significantly, higher growth rates were obtained from Egregia menziesii, M. pyrifera and P. perforata. Feed conversion ratio ranged from 6.5 to 42.4 for P. perforata and Ulva sp. Protein, carbohydrates and lipid contents in the digestive gland ranged from 113 to 180, 98.3 to 448 and 17.2 to 23.3 mg g^?1_, respectively. In muscle, the ranges were 66.9–123, 9.5–23.2 and 2.8–3.9 mg g^?1, respectively. This study shows that rehydrated Egregia menziesii, M. pyrifera and, particularly, P. perforata are more efficient in promoting growth than Ulva sp. and E. arborea which match results reported by other authors when using the same fresh macroalgae.     

Stocking density and Piscirickettsia salmonis infection effect on Patagonian blennie (Eleginops maclovinus,Cuvier 1830) skeletal muscle intermediate metabolism

The need to expand aquaculture production has led to other fish to be considered as potential species for culture, such as the sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1830). The aim of this study was to determine the cumulative effect of density and pathogen infection by protein extract of Piscirickettsia salmonis on skeletal muscle metabolism. In a first experiment, specimens were submitted to three different stocking densities: (1) 3.1 kg m^?3, (2) 15 kg m^?3 and (3) 60 kg m^?3, for a period of 10 days. In a second experiment, metabolic changes caused by an infection of P. salmonis protein extract (a single injection of 0.5 μL P. salmonis protein extract g body weight^?1 was inoculated in the fish) and its combined effect with stocking density was assessed during a period of 10 days. This study concludes that stress caused by high stocking density led to the reorganization of some metabolic routes to fulfill skeletal muscle energy needs. Furthermore, infection response by pathogen P. salmonis differed when stocking density increased, suggesting an increase of energy needs with density in skeletal muscle of infected fish.     

Autocatalytic pathways to cell death: A new analysis of the tuna burn problem

During capture and storage of tuna, a small but significant number of fish display a characteristic muscle degeneration termed tuna burn. Based on detailed amino acid analyses and on previous studies of metabolite changes during online swimming of tuna, a new model of the etiology of burnt muscle is developed. According to this model oxygen-lack to white muscle (developing initially during capture) leads to a metabolic collapse, to a drop in ATP concentration, to a consequent opening of ATP-dependent K⁺ channels, with an efflux of K⁺, and thus to a collapse of membrane potential. When the membrane potential falls far enough to open voltage-dependent Ca⁺⁺ channels, Ca⁺⁺ influx occurs leading to elevated Ca⁺⁺ concentrations in the cytosol. This process is augmented by simultaneous movement of Ca⁺⁺ from sarcoplasmic reticulum (SR) and from mitochondria into the cytosol. At high intracellular concentrations Ca⁺⁺ can be devastating. One of its more notable effects involves the activation of Ca⁺⁺-dependent proteases, which preferentially target key components of the contractile machinery (troponins, tropomyosin, C-protein, M-protein, Z-discs, -actinin) and thus cause disassembly of myofilaments prior to any significant hydrolysis of myosin or actin. This process is autocatalytic in the sense that Ca⁺⁺-activated proteases may act upon SR, thus increasing Na⁺ /Ca⁺⁺ exchange, and ultimately adding more Ca⁺⁺ to the cytosolic pool. According to this model, the difference between burnt and unburnt regions of the myotome is simply due to how far each region has moved along this self-destructive, autocatalytic pathway. The model is helpful in explaining previously perplexing data and in making useful (i.e. measurable) predictions for further studies of this important problem.     

Amino acid sequences of α-skeletal muscle actin isoforms in two species of rattail fish, Coryphaenoides acrolepis and Coryphaenoides cinereus

SUMMARY: The cDNA clones of α-skeletal actins were isolated from the skeletal muscle of two species of rattail fish, Coryphaenoides acrolepis and Coryphaenoides cinereus . The complete nucleotide sequences of the cDNA and their deduced amino acid sequences were determined. Each of the two species had two α-skeletal actin cDNA. The nucleotide sequences of the coding region of the two α-skeletal actin isoform genes within each species had 92.0 and 91.8% homology. From the cDNA sequences of the four α-skeletal actin isoforms in the two species, amino acid sequences of 377 amino acid residues were deduced. It was predicted that the two N-terminal amino acid residues of each protein are processed after translation. The amino acid sequences of α-skeletal actin 1 in the two Coryphaenoides species were identical, as were the amino acid sequences of α-skeletal actin 2 in the two species. The amino acid sequences of the two α-actin isoforms, α-skeletal actin 1 and α-skeletal actin 2, differed by only a single amino acid, Ala/Ser at the 155th position. Northern blot analysis showed that a similar amount of each of the two α-actin isoform mRNA was expressed in the skeletal muscle of the two Coryphaenoides species.     

Structural and thermodynamic characterization of tropomyosin from fast skeletal muscle of bluefin tuna

ABSTRACT:   Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7–98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.     

Specific binding of [3H]17α,20β-dihydroxy-4-pregnen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss)

Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to plasma membranes prepared from defolliculated oocytes of rainbow trout (Onchorhynchus mykiss) was identified and characterized. Binding was rapid and reached equilibrium in 30 min. 17α,20β-DP strongly inhibited [³H] 17α,20β-DP binding in a competitive manner. Scatchard analysis revealed two different binding sites: a high affinity binding site with a K_d of 18 nM and a B_max of 0.2 pmoles/mg protein; and a low affinity binding site with a K_d of 0.5 μM and a B_max of 1 pmoles/mg protein. This binding activity was successfully solubilized with n-heptyl-β-D-thioglucoside. [³H]17α,20β-DP binding to solubilized preparations reached equilibrium in 1h, and was competitively inhibited with 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one. However, Scatchard analysis showed a single binding site with a K_d of 0.3 μM. The reason for the disappearance of the high affinity binding site in solubilized preparations remains unclear. These results demonstrate that a specific binding site for 17α,20β-DP exists in the plasma membrane of rainbow trout oocytes.

---

Résumé Une liaison spécifique de le [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), avec des membranes plasmiques d'ovocytes défollicularisés de truite arc-en-ciel (Onchorhynchus mykiss), a été identifiée et caractérisée. Sa cinétique est rapide et atteint son équilibre en 30 minutes. Le 17α,20β-DP inhibe fortement, et de manière compétitive, la liaison de la [³H] 17α,20β-DP. Une étude de Scatchard a mis en évidence deux sites diffŕents de liaison: un site de forte affinité, de K_d 18 nM et de B_max 0,2 pmoles/mg de protéine; et un site de faible affinité, de K_d 0,5 μM et de B_max 1 pmoles/mg de protéine. L'activité de liaison a été solubilisée, avec succés, par le n-heptyl-β-D-thioglucoside. Dans la fraction soluble, la liaison de le [³H]17α,20β-DP atteint un équilibre en 1h.; et elle est complétement inhibiée par la 17α,20β-DP et le 17α,20β,21-trihydroxy-4-pregnen-3-one. Cependant, une étude de Scatchard ne permet de déceler qu'un seul site de liaison, de K_d 0,3 μM. La disparition du site de liaison de forte affinité dans la fraction soluble reste inexpliquée. Ces résultats démontrent l'existence d'un site spécifique de liaison du 17α,20β-DP dans les membranes plasmiques des ovocytes de truite arc-en-ciel.

     

Endocrine stress response and abnormal development in carp (Cyprinus carpio) larvae after exposure of the embryos to PCB 126

To investigate whether PCB 126 exposure duringembryonic development induces an endocrine stressresponse in larval carp, eggs were exposed,containing 0.01% ethanol (vehicle-control), 10^-11,immediately after fertilization, for 48 h to water10^-10 or 10^-9 mol l^-1 PCB in 0.01% ethanol. Eggsincubated in water served as controls. After transferto PCB-free water, mortality, the incidence ofyolk-sac and pe-ricardial oedema, wet and dry weight,rate of skin pigmentation, and whole-body contents ofthe stress hormones ACTH, -MSH and cortisol weredetermined at 48, 96, 144, 168, 192 and 216 hpost-fertilization. Except for the dry weight, allparameters of animals exposed to 10^-10 and 10^-9 moll^-1 PCB increased in a concentration-related manner.However, these changes became evident only at 144 hpost-fertilization, i.e. after resorption of theyolk-sac. Swelling of the yolk sac and pericardiumoccurred, and whole-body ACTH, -MSH and cortisollevels increased. Although animals exposed to 10^-10and 10^-9 mol l^-1 PCB displayed stable but elevatedwhole-body ACTH and -MSH levels until 216 h,whole-body cortisol concentrations gradually decreasedfrom 168 h post-fertilization, and were significantlybelow control values at 216 h post-fertilization.Exposure of the carp embryos to 10^-11 mol l^-1 PCB only increased whole-body -MSH levels. Increased whole-body ACTH and cortisol levels indicate that PCBinduces a stress response in carp larvae, possiblymediated by a disturbed hydromineral balance (oedema).We further suggest that the PCB-stimulated bodypigmentation is mediated by a stimulation of -MSHsecretion.     

Stimulation of insulin-like growth factor-I production by recombinant bovine growth hormone in Mozambique tilapia, Oreochromis mossambicus

We have previously reported growth-promoting effects of recombinant bovine growth hormone (rbGH) in Mozambique tilapia, Oreochromis mossambicus, after 4 weekly injections or a single injection of slow-releasing formulation (Posilac^®) (Leedom et al. 2002). In order to obtain further understanding of the role of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis in growth in the tilapia, the effects of rbGH on plasma and mRNA levels of IGF-I were examined. Plasma IGF-I levels were significantly increased after rbGH and Posilac^® injections, and a significant correlation was observed between plasma IGF-I levels, body length and mass in both treatments. IGF-I mRNA levels in the liver and in the skeletal muscle were also significantly increased after rbGH and Posilac^® injections, indicating that IGF-I gene expression in these tissues is under control of circulating GH. IGF-I mRNA levels in the gill were not affected by treatment. Liver IGF-I mRNA levels were significantly correlated with body length and with body mass after rbGH and Posilac^® injections. These results indicate that the growth-promoting effect of rbGH in this species is mediated to a significant extent via its stimulation of hepatic production of IGF-I and the resulting increase in plasma IGF-I, and also possibly through locally produced IGF-I in the skeletal muscle, acting in a paracrine or autocrine fashion.     

Recovery from nitrite-induced methaemoglobinaemia and potassium balance disturbances in carp

The ability of carp to recover from nitrite-induced methaemoglobinaemia and disturbances in potassium balance and cell volume was studiedin vivo andin vitro. Nitrite accumulated to a plasma concentration of 3 mM during 2 days of nitrite exposure was eliminated from the plasma within 2–3 days in clean water. The nitrite-induced methaemoglobinaemia disappeared after 3 days of recovery. During nitrite exposure, K⁺ was lost from the red blood cells (RBCs) and from skeletal muscle tissue, which led to reduced cell volume and an extracellular hyperkalaemia. Extracellular [K⁺] rose less than predicted if lost K⁺ had remained in the extracellular space, suggesting further transport of K⁺ to the environment. The intracellular K⁺ and water content were restored after few days of recovery in clean water, but this was paralleled by development of an extracellular hypokalaemia. This shows that intracellular K⁺ balance was reestablished at the expense of the extracellular compartment, and supports that an overall K⁺ deficit resulted from K⁺ loss to the environment during nitrite exposure. Ventricle tissue differed from skeletal muscle and RBCs by not loosing K⁺ and by having increased sodium and water contents during nitrite exposure. These changes were corrected by recovery in nitrite-free water. In vitro addition of nitrite to blood with low O₂ saturation induced metHb formation and RBC K⁺ efflux. Subsequent reduction of metHb to functional Hb was similar in blood with low and high O₂ tension. A net re-uptake of K⁺ was observed only in RBCs with low O₂ saturation and when metHb reached low values.     

Effects of dietary choline supplementation on growth performance,lipid deposition and intestinal enzyme activities of blunt snout bream Megalobrama amblycephal fed high‐lipid diet

This study aimed to investigate the effects of dietary choline supplementation on growth, lipid deposition and intestinal enzyme activities of Megalobrama amblycephala. Fish were fed four diets with two lipid levels (50 and 150 g kg^?1) and two choline supplementations (600 and 1600 mg kg^?1) for 8 weeks. Feed conversion ratio (FCR), viscerosomatic index (VSI), hepatosomatic index (HSI), intraperitoneal fat (IPF) ratio, whole‐body and muscle lipid contents, intestinal lipase activities and lipoprotein lipase (LPL) activities all increased significantly (P < 0.05) as lipid levels increased, whereas the opposite was true for whole‐body and muscle moisture contents and intestinal amylase activities. VSI, IPF ratio and whole‐body lipid contents all decreased significantly (P < 0.05) with increasing dietary choline supplementations. Weight gain, muscle moisture content all increased significantly (P < 0.05) with increasing dietary choline supplementations when dietary lipid levels reached 150 g kg^?1, whereas the opposite was true for FCR, IPF ratio, IPF and liver LPL activities. In addition, abnormal hepatocytes were found in the liver of fish fed 150 g kg^?1 lipid with 600 mg kg^?1 choline supplementation. The result of this study indicated that extra choline supplementation can improve growth performance, intestinal enzymes activities and reduce excessive lipid deposition of M. amblycephala fed high lipid.     

In vivo activity of native GnRHs and their analogues on ovulation in the African catfish, Clarias gariepinus (Burchell)

Four distinct forms of native gonadotropin‐releasing hormone (GnRH) and two newly designed analogues were tested for their in vivo activity to induce ovulation in African catfish. The effects of these peptides on ovulatory parameters were compared with those of carp pituitary and [d ‐Ala⁶, Pro⁹‐NEt]‐mammalian GnRH analogue (mGnRHa), two tested ovulation‐inducing agents in African catfish. Assessment of ovulation was carried out by determining the ovulation ratio and the relative quantity of egg produced. From the results of the experiments, the order of potency of the native GnRH peptides is summarized as chicken GnRH‐II (cGnRH‐II) >salmon GnRH (sGnRH) >mammalian GnRH >chicken GnRH‐I (cGnRH‐I). Chicken GnRH‐II was as potent as mGnRHa while cGnRH‐I was totally ineffective. The new d ‐Orn⁶‐cGnRH‐II and d ‐Orn⁶‐sGnRH with a substitution at position 6 with d ‐isomer residue were as potent as the most extensively used mGnRHa, indicating that the position 6 modification might be more crucial than the substitution at the C‐terminal. On the basis of our results, the potential use and incorporation of cGnRH‐II and sGnRH for the development of more generic spawning induction therapies are suggested.     

Comparative performance of growth,vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus

Growth, skeletal structure and muscle composition of cold‐shock‐induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X‐ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one‐sided compression, two‐sided compression and vertically shifted. The trunk region (V8–18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.     

Toxicity of cultured puffer fish and seasonal variations in China

To confirm the toxicity or the tetrodotoxin (TTX) content of cultured puffer fish in China, 565 specimens of cultured puffer fish (Takifugu rubripes, Takifugu fasciatus or Takifugu obscurus, Takifugu flavidus and Takifugu bimaculatus) were collected from five provinces from November 2007 to November 2008. The toxicity of the skin, muscle, liver and testis/ovary was examined using methods based on a mouse assay for TTX. The results indicated that the skin, muscle, liver and testis of specimens were ‘non‐toxic’ in both of the mouse assays [<10 mouse unit (MU) g^?1 or 2.2 μg g^?1] from the viewpoint of food hygiene. However, TTX was detected from an ovary sample of the cultured T. rubripes collected from the Shandong province in March 2008, which was 125.43 MU g^?1 tissue. The average TTX contents in the muscle, liver and skin of T. rubripes from Hebei and Shandong provinces were the highest in May or July (1.31, 0.90 and 0.62 MU g^?1, respectively, in Hebei, and 1.57, 0.32 and 0.16 MU g^?1, respectively, in Shandong) and the lowest in January (<0.1 MU g^?1 or 0.02 μg g^?1). Thus, non‐toxic cultured puffer fish could be produced successfully in mainland China.     

A NMR-based metabolomic approach for differentiation of hagfish dental and somatic skeletal muscles

The hagfish dental muscle is a large and specialized element of the feeding apparatus that helps ingest food. This muscle has enzymatic activities and contractile properties different from the hagfish somatic skeletal muscle. To verify the functional relevance of protein alterations, we examined the metabolomic differentiation of hagfish dental and somatic skeletal muscles using ¹H-nuclear magnetic resonance (NMR)-based metabolomics and multivariate analysis that separated hagfish dental and somatic muscles by principal component analysis and partial least squares for discriminant analysis. Our analysis of assigned metabolites showed that anserine and taurine levels were higher in dental muscle, but creatine, fructose, glucose, glycerate, pyruvate, and succinate levels were higher in somatic muscle. We concluded that the primary energy sources of dental and somatic muscles are related to the citric acid cycle and the anaerobic glycolysis and metabolism of creatine. Thus, ¹H-NMR-based metabolomics can be integrated with the previous proteomic approach to derive biochemical and physiological information about hagfish muscles.     

Ion levels in the gastrointestinal tract content and plasma of four teleosts with different feeding habits

The levels of various ions (Na⁺, Cl⁻, Ca²⁺, Mg²⁺, K⁺) in the fluid phase of the gastrointestinal contents and of the plasma were determined in four teleosts with different feeding habits: traira (Hoplias malabaricus), hassar (Hoplosternum littorale), silver catfish (Rhamdia quelen), and grass carp (Ctenopharyngodon idella). The results showed that most ionic levels in the fluid phase of the gastrointestinal tract seem to be affected by feeding habit and that these levels can be different from those found in the plasma. In practical terms, these results suggest that ionic levels of the gastrointestinal contents should be considered when preparing solutions for studies of intestinal absorption in fishes.