A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila.

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.     

Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.     

Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity.

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.     

The effects of temperature- and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain microsomes

Brain microsome phospholipids and their acyl groups, from temperature and oxygen acclimated goldfish, were investigated. At the lower acclimation temperature (5C) the proportion of ethanolamine- to choline-glycerophosphatides (GPE/GPC) was increased, and the proportion of phosphatidal ethanolamine value decreased. A rise in the n-6/n-3 fatty acyl group also occurred in cold acclimation. Irrespective of acclimation temperature, 25 degrees C or 5 degrees C, a partial replacement of GPC by GPE occurred when the concentration of oxygen was increased; conversely the GPE/GPC ratio decreased at the hypoxic level. The plasmalogen GPE content increased as the oxygen concentration was raised. A rise in the n-6/n-3 ratio, for ethanolamine glycerophosphatides and phosphatidyl choline, occurred when the oxygen concentration was increased (hypoxia to hyperoxia). It is concluded that the lipid alterations associated with thermal acclimation are, in part, attributable to the concomitant change in oxygen concentration.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

Nucleotide pool and the proliferative process: an NMR study of the liver regeneration

The metabolic changes occurring during liver regeneration after partial hepatectomy have been followed by examination of the perchloric extracts by means of NMR spectroscopy. Proton spectra show an abrupt decrease of glycogen, glucose and nucleotides, which are essentially ribonucleotides, in the first hours after hepatectomy. Nucleotides begin to store up again in the third day after hepatectomy, while glucose and glycogen storage builds up from the second day. 31P data evidence a sharp drop of phosphomonoesters, i.e. monophosphosugars (including AMP) and phosphocholine, and phosphodiesters, i.e. GPC and GPE, soon after hepatectomy and a recovery of the initial levels approximately at the 60th hour and a further increase of PDE at later times. The NMR findings are in agreement with the biochemical knowledge of the course of liver regeneration.     

A new experimental approach and signal processing scheme for the detection and quantitation of ³¹P brain neurochemicals from in vivo MRS studies using dual tuned (¹H/³¹P) head coil

Brain (31)P-neurometabolites play an important role in energy and membrane metabolism. Unambiguous identification and quantification of these neurochemicals in different brain regions would be a great aid in advancing the understanding of metabolic processes in the nervous system. Phosphomonoester (PME), consisting of phosphoethanolamine (PE) and phosphocholine (PC), is the "building block" for membranes, while phosphodiesters (PDE), consisting of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) metabolites are involved in the membrane breakdown process. In the clinical setting, generating well-resolved spectra for PC, PE, GPC, and GPE could be crucial phospholipids in providing information regarding membrane metabolism. We present here a new experimental approach for generating well-resolved (31)P spectra for PC and PE as well as for GPC, GPE, and other (31)P metabolites. Our results (based on uni-dimensional (1D) and multi-voxel (31)P studies) indicate that an intermediate excitation pulse angle (35°) is best suited to obtain well-resolved PC/PE and GPC/GPE resonance peaks. Our novel signal processing scheme allows generating metabolite maps of different phospholipids include PC/PE and GPC/GPE using the 'time-domain-frequency-domain' method as referred to in the MATLAB programming language.     

A new experimental approach and signal processing scheme for the detection and quantitation of P brain neurochemicals from in vivo MRS studies using dual tuned (H/P) head coil

Brain ³¹P-neurometabolites play an important role in energy and membrane metabolism. Unambiguous identification and quantification of these neurochemicals in different brain regions would be a great aid in advancing the understanding of metabolic processes in the nervous system. Phosphomonoester (PME), consisting of phosphoethanolamine (PE) and phosphocholine (PC), is the “building block” for membranes, while phosphodiesters (PDE), consisting of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) metabolites are involved in the membrane breakdown process. In the clinical setting, generating well-resolved spectra for PC, PE, GPC, and GPE could be crucial phospholipids in providing information regarding membrane metabolism. We present here a new experimental approach for generating well-resolved ³¹P spectra for PC and PE as well as for GPC, GPE, and other ³¹P metabolites. Our results (based on uni-dimensional (1D) and multi-voxel ³¹P studies) indicate that an intermediate excitation pulse angle (35°) is best suited to obtain well-resolved PC/PE and GPC/GPE resonance peaks. Our novel signal processing scheme allows generating metabolite maps of different phospholipids include PC/PE and GPC/GPE using the ‘time-domain–frequency-domain’ method as referred to in the MATLAB programming language.     

Renal water and solute excretion in the Atlantic stingray in fresh water

Freshwater, male Atlantic stingrays Dasyatis sabina , from Lake Jesup, Florida, U·S·A·, excreted a dilute urine similar in composition to freshwater teleosts and lampreys with the exception that urea was the primary osmolyte. Urine flow rate was 2·5 to 10 fold higher than that reported for freshwater teleosts resulting in high free-water clearance. Mass-specific free-water clearance values from euryhaline elasmobranchs inhabiting freshwater environments greatly exceed those for freshwater teleosts and are nearly equivalent to those of freshwater lampreys.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).     

Exploration du plasma seminal humain: Nouvelle approche par la resonance magnetique nucleaire du proton (RMN-1H)

Human seminal plasma glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citric acid (Cit) and lactic acid (Lac) were analzed by measuring the peak area of proton nuclear magnetic resonance (NMR) spectra in four groups of patients: 21 men with secretory azoospermia; 14 men with excretory azoospermia after vasectomy; seven patients presenting with very severe oligoasthenozoospermia; and 18 normozoospermic subjects. When comparing secretory and excretory azoospermia cases with normozoospermic men, seminal plasma GPC, Cit and Lac peak areas were lower in azoospermic cases compared to normozoospermic men (16.79, 8.18 and 2.28 versus 23.38, 10.58 and 4.3. arbitrary units respectively, P <0.01). The peak area ratios Cit/Lac and GPC/lac were significantly different between normozoospermic men and either secretory or excretory azoospermic groups (P<0.01). A significant difference was also found in the GPE/GPC peak intensity ratio between secretory and excretory azoospermia cases (P<0.001). These results demonstrate that quantitative markers such as GPC, GPE, Cit and Lac may be useful in the examination of human seminal plasma by proton NMR in infertility investigations.     

Chemical assessment of phospholipid and phosphoenergetic metabolites in regenerating rat liver measured by in vivo and in vitro 31P-NMR.

For the assessment of 31P-NMR spectroscopic data, phospholipid precursors (phosphorylethanolamine (PE) and phosphocholine) and catabolites (glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC)), as well as adenosine phosphates were chemically determined in regenerating rat liver. The data were compared with those obtained by in vivo and in vitro 31P-NMR spectroscopies. Chemical assay revealed a significant increase of PE and a decrease of GPE, GPC and ATP in hepatectomy group compared to sham operation group. The values obtained by in vitro NMR were in good agreements with those of chemical assay, but significant differences between the two groups were observed only in PE and inorganic phosphate (Pi). Noticeable increase in PME was not detected by in vivo 31P-NMR spectroscopy, although the increase of PE was about 2.5-times that of the control and its constitution ratio to the whole phosphomonoester (PME) was less than 15%. On the other hand, in vivo NMR showed a large phosphodiester (PDE) peak occupying approx. 40% of the total phosphorus signal, while the contribution of its constituents, GPE and GPC was about 5% found by both chemical assay and in vitro NMR. The PDE peak in in vivo NMR seemed to reflect the membrane phospholipid itself rather than its catabolites. A slight decrease of phosphoenergetic level in regenerating rat-liver was commonly suggested by all three analytical methods.     

Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy,immunohistochemistry and radioimmunoassay

Summary The immunoreactivity of atrial natriuretic peptide and ultrastructure of cardiocytes were examined in 5 species each of freshwater and seawater teleosts, as well as in 2 species each of elasmobranchs and cyclostomes. Immunoreactivity was strong in the atria of Cyprinus carpio, Anguilla japonica and Conger myriaster, rather weak in atria of Channa maculata, Lepomis macrochirus, Salmo gairdneri, Oplegnathus fasciatus and Eptatretus burgeri, very weak in atria of Pagrus major, Trachurus japonicus and Triakis scyllia, and not detectable in atria of Hexagrammos otakii, Narke japonica and Lampetra japonica. The immunoreactivity of the atrial cardiocytes was generally stronger in freshwater than seawater fish. Ventricular immunoreactivity was detected only in 7 species, always being weaker than that observed in the atrium. Ultrastructurally, however, secretory granules were found in atria and ventricles of all species examined, being more frequent in the former than the latter. By radioimmunoassay, immunoreactive ANP was detected in the extracts of blood plasma and both atrial and ventricular tissues of all species examined. There were no statistically significant differences in the values between freshwater and seawater species.     

The effect of temperature-and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain mitochondria

Goldfish (Carassius auratus L.) were temperature- and oxygen-acclimated and the composition of the phospholipids and their acyl groups in brain mitochondria was determined. The proportion of ethanolamine to choline phospholipid was greater while the plasmenyl ethanolamine value (P-GPE/D- + P-GPE) was lower at the low acclimation temperature. For the ethanolamine glycerophospholipids, a rise in the ratio n-6/n-3 fatty acyl groups occurred with cold acclimation. No significant change in the ratio was exhibited by phosphatidyl choline. When the oxygen level was increased, at either acclimation temperature, a rise in the GPE/GPC ratio and the plasmenyl ethanolamine value resulted. The n-6/n-3 ratio was generally increased for the ethanolamine classes when the oxygen concentration was raised. The possible significance of these changes is discussed.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions

Although prolactin has been demonstrated to be the main hormone involved in adaptation to dilute media in several freshwater teleosts, few studies have been conducted in marine teleosts. In the Mediterranean, the sea bass Dicentrarchus labrax inhabits environments ranging from the open sea to coastal lagoons, where salinity varies greatly. We characterised the prolactin (prl) gene and analysed its expression in two organs (gill and intestine) in D. labrax acclimated to either freshwater or seawater. A 2819 bp long sequence encompassing the prl gene and a part (282 bp) of the promoter were identified, and these comprised 5 coding exons separated by 4 introns. Prolactin was similarly expressed in fresh- and seawater adapted fish, although expression in gills was significantly greater than in the intestine. Nonetheless, individuals unable to successfully regulate osmotic balance in freshwater presented overall low expression rates. Results are discussed according to the mechanism of sea bass adaptation in the wild and to their life cycle between open sea and lagoons. Finally, a phylogenetic analysis indicated that teleosts are not branched according to their life-history features (e.g. seawater vs. freshwater habitats), and no signature of positive selection was detected across the phylogeny of the prl gene in teleosts.     

Variations in renal corpuscular morphology with adaptation to sea water in the rainbow trout, Salmo gairdneri Richardson

The kidneys of similar sized rainbow trout from freshwater and seawater environments were examined and several parameters of renal corpuscular morphology were measured, The glomeruli of the seawater trout were significantly smaller and contained significantly fewer red blood cells than those of the freshwater fish. There was also some evidence of a decreased number of renal corpuscles per unit area, and an increased proportion with no apparent capsular space in the seawater trout.     

Arsenobetaine in Atlantic salmon (Salmo salar L.): influence of seawater adaptation

Glycine betaine has been suggested to improve the maintenance of ionic and osmotic homeostasis during seawater adaptation in teleost fish. Arsenobetaine may also behave as an osmolyte, due to its structural similarity to glycine betaine. The influence of seawater adaptation on intestinal uptake and muscle accumulation of arsenobetaine in the teleost Atlantic salmon (Salmo salar L.) was investigated. Atlantic salmon (freshwater and seawater adapted) were given a single oral dose of arsenobetaine, which was absorbed over the intestine within 6 h after exposure. Seawater adapted Atlantic salmon had significantly higher levels of accumulated arsenobetaine in blood compared to the freshwater adapted salmon. However, seawater adaptation had no effect on the levels of accumulated arsenobetaine in muscle tissue. Similar retention of the administered dose was found in muscle tissue in both freshwater and seawater adapted salmon, with 49+/-6% and 50+/-10% retention after 144 h, respectively. Results indicate that muscle retention was not influenced by salinity in seawater adapting teleosts.     

Ureogenesis in a freshwater teleost: an unusual sub-cellular localization of ornithine-urea cycle enzymes in the freshwater air-breathing teleost Heteropneustes fossilis.

Sub-cellular localization of different ornithine-urea cycle enzymes was studied in the liver and kidney of a freshwater air-breathing teleost. Carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase were found to be localized inside the mitochondria, and argininosuccinate synthetase and argininosuccinate lyase were found in the soluble fraction. Mitochondrial localization of arginase, a feature known in marine elasmobranchs and toadfishes, indicates the evolutionary position of H. fossilis to be different from that of present day freshwater teleosts.