Mitochondrial NAD(P)-dependent malic enzyme from herring testicular tissue: Purification,kinetic behaviour and regulatory properties

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD⁺ oxidoreductase (decarboxylating)] was purified from herring testicular tissue to a specific activity of 26.4 μmol NADH/min/mg protein. Herring testicular tissue is one of the most abundant sources of this enzyme. The purification procedure involved differential centrifugation of mitochondria and then chromatography on DEAE-Sephacel, Red Agarose and Sephacryl S-300. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of Mn²⁺ and either NAD or NADP. Under V_max conditions the ratios for the rate of NAD/NADP reduction was 1.8. A study of the reductive carboxulation reaction indicated that this enzyme reaction is reversible; at pH 7.0 the reverse reaction exhibited 22% of the activity of forward reaction. Some kinetic characteristics of the enzyme were determined. ATP was found to be a competitive inhibitor with respect to malate. Fumarate reversed ATP inhibition. Regulation of NAD(P)-dependent malic enzyme from herring testicular tissue mitochondria could respond to changing levels of mitochondrial ATP and fumarate in vivo.     

Assessing the effects of line capture and barotrauma relief procedures on post-release survival of key tropical reef fish species in Australia using recreational tagging clubs

Abstract  Common coral trout, Plectropomus leopardus Lacepède, crimson snapper, Lutjanus erythropterus Bloch, saddletail snapper, Lutjanus malabaricus (Bloch & Schneider), red emperor, Lutjanus sebae (Cuvier), redthroat emperor, Lethrinus miniatus (Schneider) and grass emperor, Lethrinus laticaudis Alleyne & Macleay, were tagged to determine the effects of barotrauma relief procedures (weighted shot-line release and venting using a hollow needle) and other factors on survival. Release condition was the most significant factor affecting the subsequent recapture rate of all species. Capture depth was significant in all species apart from L. malabaricus and L. miniatus , the general trend being reduced recapture probability with increasing capture depth. Recapture rates of fish hooked in either the lip or mouth were generally significantly higher than for those hooked in the throat or gut. Statistically significant benefit from treating fish for barotrauma was found in only L. malabaricus , but the lack of any negative effects of treating fish indicated that the practices of venting and shot-lining should not be discouraged by fisheries managers for these species.     

Effects of dietary selenium on growth performance and oxidative stress in juvenile grass carp Ctenopharyngodon idellus

An 8‐week feeding trial was conducted to investigate the effect of dietary selenium (Se) on feed intake, weight gain and antioxidant activity in juvenile grass carp (11.2 ± 0.03 g). Six Se levels (0.13, 0.41, 0.56, 1.12, 2.18 and 4.31 mg/kg) of semi‐purified diets were assayed in triplicate. The maximum weight gain, specific growth rate and feed intake were obtained in fish fed with 1.12 mg Se/kg diet. Hepatic glutathione peroxidase activity was markedly increased when dietary Se ≤1.12 mg/kg diet and reached a plateau when dietary Se ≥1.12 mg/kg diet. Hepatic superoxide dismutase and serum catalase activities in juvenile grass carp fed with 0.56, 1.12 and 2.18 mg Se/kg diets were all significantly higher than those in the other groups. The malondialdehyde content in liver and serum was firstly decreased and then increased with increasing dietary Se content, and the lowest content was observed in fish fed with 1.12 mg Se/kg diet. With the increase in Se level, the activities of serum alanine aminotransferase and aspartate aminotransferase were reduced. In addition, serum alkaline phosphatase activity and albumin content were highest in fish fed with 1.12 mg Se/kg diet. This study indicated that both the Se deficiency and excess of Se caused negative effect on the oxidative stress in juvenile grass carp and suggested that the health‐giving concentration of dietary inorganic Se was 1.12 mg/kg diet. Moreover, based on the broken‐line regression analysis of weight gain, the optimal concentration of dietary inorganic Se was 0.83 mg/kg for juvenile grass carp.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

Effects of temperature and lipid droplet adherence on mortality of hatchery-reared southern hake Merluccius australis larvae

Effects of exogenous (water temperature) and endogenous (lipid droplet adherence) factors were experimentally tested on early survival of southern hake Merluccius australis reared under controlled conditions. Experiments to determine the effect of temperature (10, 12 and 14 °C) on larval growth rates and yolk-sac absorption rates of unfed southern hake were carried out under laboratory conditions. There was no significant differences in growth rates at the temperature range tested (ANCOVA, F = 0.164, p > 0.25), but yolk-sac absorption rates and mortality increased with temperature (ANCOVA, F = 53.84, p < 0.001). A high percentage (between 31 and 81%) of hake eggs showed a lipid droplet not adhered (i.e., freely moving in the yolk, and not located in the posteriormost portion of the yolk-sac). In a second experiment, fed southern hake larvae with the lipid droplet not adhered during embryonic development did not survive after yolk-sac absorption. This study provides the first data on the influence of the lipid droplet absorption on larval survival of cultured hake, and can be used as an early indication of the quality of the batch.     

Guanine-cytosine contents of the host and symbiont cDNA in a symbiotic coral

ABSTRACT:   Hermatypic (reef-building) corals harbor dinoflagellate endo-symbionts Symbiodinium spp. In studying gene expression in such symbiotic corals, problems arise regarding how to distinguish the coral and symbiont mRNA, and how to estimate their fractions in the mRNA population of the holobiont (symbiotic complex of the coral and Symbiodinium cells). In this study, these issues were addressed using juveniles of hermatypic coral Acropora tenuis in symbiosis with Symbiodinium cells of strain PL-TS-1. First, the guanine-cytosine (GC) contents were determined in expressed sequence tags (EST) from PL-TS-1 cells cultured in vitro and symbiont-free larvae of A. tenuis , and their average GC contents were found to be significantly different. The average GC content of the EST from the holobiont was much closer to that of A. tenuis larvae, suggesting that the majority (>90%) of mRNA isolated from the holobiont originated in the host. In protein-coding sequences, little overlap was observed between the GC-content distributions of PL-TS-1 cells and A. tenuis larvae. All of the coding sequences ( n  = 59) found in the A. tenuis EST had GC contents below 0.5, whereas the GC content exceeded 0.5 in the majority (43/44) of coding sequences from the nuclear genome of PL-TS-1 cells.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

Effect of bleeding on the quality of amberjack Seriola dumerili and red sea bream Pagrus major muscle tissues during iced storage

The effect of bleeding on white muscle quality in amberjack and red sea bream was evaluated by measuring ATP-related compounds, volatile basic nitrogen (VBN), and the trimethylamine (TMA) content. The freshness was evaluated by the K value, and the degree of spoilage was elucidated by VBN and TMA. ATP was rapidly converted to ADP and AMP in the muscle and IMP was the main product of ATP degradation during iced storage. For both species, the IMP content was higher in the muscles of fish that were bled than in those unbled during iced storage. Conversely, the K value and the levels of hypoxanthine (Hx) and VBN were higher in the muscle tissue of unbled fish than in the bled tissue of both species. Similarly, the TMA content was higher in the unbled muscle tissues of both species after a week of storage.     

Mycoplasma contamination in fish cell lines: an evaluation of detection by differential incorporation of 3H-uridine and 14C-uracil

Abstract. Differential incorporation of uridine and uracil was used to assay for mycoplasma contamination in five fish cell lines: bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The method was not suitable for monitoring BF-2, CHSE-214, FHM, and RTG-2 cell lines because they incorporated uracil. Differential incorporation of uridine and uracil may be applicable for screening EPC cells because only this cell line could distinguish cultures experimentally infected with Mycoplasma orale from cultures known to be free from microbial contaminants.