Expression of taurine transporter in response to hypo-osmotic stress in the mantle of Mediterranean blue mussel

Expression of taurine transporter in response to osmotic stress was investigated at the protein level in the mantle of the Mediterranean blue mussel by using the specific antibody raised against the carboxy-terminal region of the deduced amino acid sequence of mussel taurine transporter. Immunohistochemical observation revealed that taurine transporter was expressed in the mantle and the expression was up-regulated in response to hypo-osmotic stress, while down-regulated in response to hyper-osmotic stress. Western blot analysis revealed major protein bands corresponding to 62 kDa and 65 kDa. In response to hypo-osmotic stress, the 62 kDa band became more intense, while it became less intense when the ambient osmolality was elevated. These results suggested that the 62 kDa taurine transporter would be implicated in hypo-osmotic adaptation.     

Taurine transporter from the giant Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: function and expression in response to hyper- and hypo-osmotic stress

ABSTRACT:   Taurine is the primary osmolyte in marine molluscs, whose cellular osmo-conforming process is vital for environmental adaptation because of a lack of osmotic homeostasis. Here, cDNA cloning and expression, and functional analyses of taurine transporter (TAUT) from the giant Pacific oyster are reported on. The deduced amino-acid sequence of oyster TAUT (oyTAUT) showed 47–51% identity to those of vertebrate TAUT, whereas identity among the vertebrates is 78–95%. Functional analysis of oyTAUT expressed in Xenopus oocytes revealed that oyTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration, compared with vertebrate TAUT. Taken together with similar functional properties of TAUT from mussel, indicated by our previous study, it is possible that these functional features reflect the internal environment of the molluscs (i.e. higher taurine and NaCl concentrations). Oyster taurine transporter mRNA expression was induced by not only hyper-osmotic stress, similar to other TAUT, but also hypo-osmotic stress. It is speculated that the expression in response to hypo-osmotic stress was induced by a substantial decrease in tissue taurine content following the decrease in the internal osmolality.     

Effects of salinity on the growth and lipid composition of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture

The direct effects of osmotic pressure (salinity) on growth performance and lipid composition were investigated in fish cells in culture. Cell lines from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce osmotic pressures varying from 300 to 500 mOsm kg⁻¹. The growth rates of the two cell lines were affected in a similar manner by the salinity of the media with the rank order for both peak cell numbers and growth rates up to the day of peak cell number being 300 > 350 > 400 > 450 > 500 mOsm kg⁻¹. Cell death occurred in both cell lines in older cultures at all salinities with the greatest loss of viable cells in media of 300 and 350 kg⁻¹. However, there were quantitative and qualitative differences between the cell lines in their lipid metabolism in response to the salinity of the media. The lipid content expressed per cell showed a positive correlation between lipid per cell and salinity in TF cells, but this was less apparent in AS cells. The percentage of total polar lipid classes increased with increasing salinity in TF cells due mainly to graded increases in the percentages of choline phospholipids. In contrast, there were no significant differences in the proportions of polar and neutral lipid classes with salinity in AS cells. The only significant effect of salinity in AS cells was a decreased proportion of dimethylacetals in total lipid at the highest salinity. The same significant effect of salinity on dimethylacetal content of total lipid was observed in TF cells. However, in addition there was a graded decrease in the percentage of 18:2n-9 in TF cell total lipid with increasing salinity. This was accompanied by increased percentages of total n-3 and n-6 PUFA with higher proportions of both groups of PUFA at 450 and 500 compared with 300 mOsm kg⁻¹. The results show that environmental salinity, in the absence of hormonal or other physiological stimuli, has direct effects on the growth and lipid metabolism of fish cells and that these effects differ in cells from different fish species.     

Effect of salinity change on free amino acid content in Pacific oyster

ABSTRACT:   In order to identify free amino acids (FAA) that are importantas intracellular osmolytes in Crassostrea gigas , we investigatedthe change in FAA content in the mantle exposed to an abrupt decreaseor increase in salinity. In hypo-osmotic adaptation, most FAA showedremarkable and synchronous decreases from 2 to 8 h, suggestingthat the non-selective efflux of FAA was mainly responsible forthe decrease in FAA. Taurine that accounted for approximately 80% oftotal FAA content contributed most significantly to the hypo-osmoticadaptation. In hyper-osmotic adaptation, significant increases inglycine, alanine, β-alanine, proline, arginine and taurinewere observed. Of these, alanine showed an immediate increase thatis important to short-term adaptation to hyper-osmolality, whiletaurine showed a slower and substantial increase that contributesto a long-term adaptation to hyper-osmolality.     

Influence of temperature on the proliferative response of rainbow trout gonadal fibroblasts to cortisol and RU 486

The rainbow trout gonadal cell line, RTG-2, which survives temperatures from 0 to 28°C and proliferates at 5 to 26°C, responded to cortisol from 28°C to 0°C by influencing [³H]-thymidine incorporation into DNA. Over the normal temperature range of rainbow trout, 10–22°C, cortisol inhibited [³H]-thymidine incorporation. The antiglucocorticoid RU 486 had no effect on [³H]-thymidine incorporation at these temperatures and blocked the response to cortisol. Another antiglucocorticoid RU 362 also had no effect but was less effective in blocking the cortisol response. During incubation at 28°C this inhibitory response to cortisol was detected inconsistently during the first 24 h but was observed consistently during the second 24 h. At 0°C, cortisol and RU 486 had no effect during short treatments, but a 60 h exposure to either steroid stimulated [³H]-thymidine incorporation over a 48 h labelling period. These results suggest that temperature shifts between 10–22°C, do not change the direction of a response to cortisol and support the use of the upper portion (20–22°C) of the temperature range for studies on salmonid cells in culture.     

Identification of a heparin-binding,mesoderm-inducing peptide in the swim-bladder of the red seabream,Pagrus major: a probable fish fibroblast growth factor

The brain, pituitary, liver, ovary and swim-bladder of the red seabream (Pagrus major) were screened for the presence of a growth factor capable of stimulating the proliferation of BHK-21 cells, and the swimbladder extract was found to exert the most powerful activity. Swim-bladder derived growth factor (SBDGF) with heparin-binding ability was isolated by heparin-affinity and size-exclusion chromatography using heparin-Sepharose CL-6B and Superose 12, respectively. The relative molecular mass of SBDGF was 22,500 and the isoelectric point was 9.4. The growth of mammalian fibroblasts (BALB/3c 3T3, BHK-21) and endothelial cells (CPAE) was markedly stimulated by SBDGF. Its growth-promoting activity was also effective in fish fibroblasts (BF-2 and RTG-2), and was heat- and acid-labile. In the mesoderm-induction assay, explants of presumptive ectoderm dissected from Xenopus laevis blastulae (stage 9) were incubated with SBDGF. SBDGF induced ventral mesoderm in the explants, including hemocyte-like cells, mesenchyme and coelomic epithelium. These properties suggest that SBDGF is a fish fibroblast growth factor and that the swim-bladder is the source.     

Effects of salinity on the fatty acid compositions of total lipid and individual glycerophospholipid classes of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture

Cells from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce OPs varying from 300 to 500 mOsm kg^–1 and the direct effects of OP (salinity) on the fatty acid compositions of the main glycerophospholipid classes were determined. The most dramatic effects of salinity on total lipid fatty acids were observed in polyunsaturated fatty acids (PUFA) in TF cells. There was a graded decrease in the percentage of 18:2n-9, and consequently total n-9 PUFA, and concomitantly increased percentages of both total n-3 and n-6 PUFA with increasing salinity. The increased n-3 and n-6 PUFA was due to significantly increased percentages of the major fatty acids in each of these groups, namely 22:6n-3 and 20:4n-6, respectively. The reciprocal changes in n-9 PUFA and n-3/n-6 PUFA in TF cell total lipid resulted in the percentage of total PUFA not being significantly affected by changes in salinity. The graded decrease in 18:2n-9 with increasing salinity in TF cells was observed in all the major glycerophospholipids but especially PE, PI and PS. Increasing salinity resulted in graded increases in the percentages of 22:6n-3 in PE and PS in TF cells. The quantitatively greatest increase in the percentage of n-6 PUFA in TF cells occurred with 20:4n-6 in PC, PE and PL. There were less significant changes in the fatty acid compositions of glycerophospholipids in AS cells. However, the proportion of total n-3 + n-6 PUFA in PE varied reciprocally with the proportion of dimethylacetals in response to salinity. Similar reciprocal changes between fatty acids in response to salinity were also evident in the quantitatively more minor glycerophospholipids PS and Pl. In PS, the percentage of 22:6n-3 was significantly lower at 400 mOsm kg^–1 whereas the proportion of total monoenes was significantly higher at that salinity. A similar inverse relationship between total monoenes and 20:4n-6 (and, to a lesser extent total saturates) in response to salinity was noted in PI. The results show that environmental salinity, without whole-body physiological stimuli, has direct effects on the fatty acid composition of major glycerophospholipid classes in fish cells and that these effects differ in cells from different fish speciesAbbreviations ANOVA analysis of variance - BHT butylated hydroxytoluene - BSA bovine serum albumin - DMA dimethylacetals - EMEM Eagle's minimal essential medium - FCS fetal calf serum - GC gas chromatography - HBSS Hank's balanced salt solution (without Ca²⁺ and Mg²⁺) - OP osmotic pressure - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - TLC thin-layer chromatography     

Refrigerated storage and cryopreservation of sperm of red drum, Sciaenops ocellatus L.

To aid in artificial spawning of sciaenid fishes, the present authors developed techniques to collect, handle and cryopreserve sperm from red drum, Sciaenops ocellatus L. Sperm were collected by removing and slicing the testis, and adding Hanks' balanced salt solution (HBSS) or NaCl solution (each at 200-400 mOsm kg^?1) as an extender. Sperm were activated with 800 mOsm kg^?1 artificial sea water (ASW) to characterize motility. Sperm reached maximum motility (highest percentage motility observed for that sample) within 8 ± 1 s (mean ± SD) and remained at maximum motility for 33 ± 4 s. Sperm were exposed to graded osmotic pressures of ASW (8-800 mOsm kg^?1) to determine the range of osmolalities that elicited motility. Threshold activation (defined as ～10% motility) occurred at 351 ± 4 mOsm kg^?1 and complete activation occurred at 539 ± 2 mOsm kg^?1. Sperm stored at 200 mOsm kg^?1 retained motility for up to 13 days. Dimethyl sulphoxide (DMSO) was used as a cryoprotectant at concentrations ranging from 7.5% to 15% (v:v) in HBSS (200 mOsm kg^?1). There were no significant differences among post-thaw motilities of sperm cryopreserved at any concentration of DMSO. Sperm thawed on the benchtop at 21°C had lower post-thaw motility than did sperm thawed at 10, 20, 30, 40, 50 or 60°C in a water bath.