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Biomedical subjects

Patricia A Wright

Publications and source records attributed to Patricia A Wright.

12 recordsLinked to original sources
PubMed2006

Removal of the chorion before hatching results in increased movement and accelerated growth in rainbow trout (Oncorhynchus mykiss) embryos.

We investigated the effects of the chorion on movement and growth in rainbow trout (Oncorhynchus mykiss) embryos. To test if the chorion restricts movement and growth before hatching, we manually removed the chorion 3-6 days before the natural time of hatching (dechorionated) and compared movement, growth and oxygen consumption in dechorionated embryos and in embryos whose chorions remained intact until the time of hatching (chorionated). Dechorionated embryos exhibited 36 times more movement before hatching compared with intact embryos. By 10 h post-hatch there was no difference in the number of movements between the two groups. At the time of hatching [30 days post-fertilization (d.p.f.)], dechorionated embryos had a significantly greater embryonic body dry mass compared with chorionated embryos, which persisted up to 45 d.p.f. At first feeding (50 d.p.f.) there was no significant difference in embryonic body dry mass between the two groups. Dechorionated embryos had a significantly greater embryonic body protein content after hatching (32, 33 d.p.f.) compared with chorionated embryos. Despite the differences in movement and growth, there were no significant differences in oxygen consumption between chorionated and dechorionated embryos. Furthermore, there was no correlation between the number of movements and oxygen consumption in rainbow trout embryos (chorionated, dechorionated, and hatched). Taken together, the data indicate that rainbow trout embryos have the capacity to be relatively active before hatching, but that the chorion restricts or inhibits movement. Moreover, precocious activity in pre-hatch embryos is correlated with accelerated growth and higher protein content, suggesting that the exercise training effect observed in adult salmonids is also present in early developmental stages.

Animals↗
PubMed2006

Characterization of diurnal urea excretion in the mangrove killifish, Rivulus marmoratus.

An unusual characteristic of nitrogen excretion in the ammoniotelic mangrove killifish Rivulus marmoratus is that urea is excreted (J(urea)) in a distinct diurnal pattern, whereas ammonia is excreted (J(amm)) at a steady rate. In this study we tested the hypothesis that the diurnal pattern in R. marmoratus is an endogenously generated pattern that is characterized as a circadian rhythm. This hypothesis was tested by measuring J(urea) and J(amm) following manipulation of feeding or lighting regimes. The diurnal J(urea) pattern in food-deprived R. marmoratus had a 24 h periodicity under normal conditions of 12 h:12 h light:dark (12:12 L:D) with 72% more urea excreted during 12:00 h and 18:00 h. In contrast, there was no significant pattern in J(amm). Fed fish (12:12 L:D) demonstrated a diurnal pattern in both J(urea) and J(amm) with up to an eightfold increase in excretion rates compared with rates obtained from food-deprived fish. Patterns of J(urea) were free running with a 24 h period under conditions of continuous darkness (0:24 L:D). Exposure to an inverse photoperiod (12:12 D:L) resulted in entrainment of the J(urea) pattern to the new photoperiod, with the highest rates of excretion occurring during midday of the new photoperiod. In contrast to R. marmoratus, nitrogen excretion rates in the zebrafish Danio rerio remained constant over time. The results of this study show that J(urea) in R. marmoratus demonstrates the characteristics of a circadian rhythm: a 24 h periodicity, a free-running rhythm in continuous conditions, and entrainment to new photoperiods.

Ammonia↗
PubMed2006

Metabolic and neuroendocrine effects on diurnal urea excretion in the mangrove killifish Rivulus marmoratus.

In mangrove killifish Rivulus marmoratus, urea excretion (J(urea)) follows a distinct diurnal pattern with the highest rates between 12:00 h and 18:00 h. We investigated the regulating mechanisms that underlie temporal rhythms in J(urea) in R. marmoratus. We hypothesized that the daily pattern of J(urea) in R. marmoratus is (1) due to diurnal changes in urea synthesis rates and ultimately metabolic rate and/or (2) controlled by neuroendocrine messengers. Oxygen consumption and whole body urea content in R. marmoratus demonstrated a clear diurnal pattern with maximum rates for both parameters occurring at 12:00 h. A strong synchrony between diurnal patterns of oxygen consumption, whole body urea content and J(urea) implicated metabolic regulation of the diurnal J(urea) pattern. Ketanserin, a 5-HT(2) receptor antagonist, and RU-486, a cortisol receptor antagonist, were used to test the second hypothesis. Increasing antagonist concentrations of either ketanserin or RU-486 resulted in dose-dependent decreases in J(urea). Application of a single dose of either antagonist significantly decreases J(urea) for up to 12 and 6 h for ketanserin and RU-48, respectively. Repeated exposure to doses of either ketanserin or RU-486 did not abolish the diurnal pattern in J(urea); however, there was a significant decrease in the amplitude of the rates. Taken together, these findings indicate that the diurnal pattern of J(urea) in R. marmoratus are regulated by both metabolic and neuroendocrine factors. We propose that cortisol and 5-HT influence the absolute rate of urea excretion by altering the permeability of the gill membrane to urea and/or the rate of urea synthesis.

Ammonia↗
PubMed2005

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion.

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.

Ammonia↗
PubMed2005

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge.

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.

Analysis of Variance↗
PubMed2004

The giant mudskipper Periophthalmodon schlosseri facilitates active NH(4)(+) excretion by increasing acid excretion and decreasing NH(3) permeability in the skin.

Periophthalmodon schlosseri is an amphibious and obligatory air-breathing teleost, which is extremely tolerant to environmental ammonia. It actively excretes NH(4)(+) in ammonia loading conditions. For such a mechanism to operate efficaciously the fish must be able to prevent back flux of NH(3). P. schlosseri could lower the pH of 50 volumes (w/v) of 50% seawater in an artificial burrow from pH 8.2 to pH 7.4 in 1 day, and established an ambient ammonia concentration of 10 mmol l(-1) in 8 days. It could alter the rate of titratable acid efflux in response to ambient pH. The rate of net acid efflux (H(+) excretion) in P. schlosseri was pH-dependent, increasing in the order pH 6.0<7.0<8.0<8.5. Net acid flux in neutral or alkaline pH conditions was partially inhibited by bafilomycin, indicating the possible involvement of a V-type H(+)-ATPase. P. schlosseri could also increase the rate of H(+) excretion in response to the presence of ammonia in a neutral (pH 7.0) external medium. Increased H(+) excretion in P. schlosseri occurred in the head region where active excretion of NH(4)(+) took place. This would result in high concentrations of H(+) in the boundary water layer and prevent the dissociation of NH(4)(+), thus preventing a back flux of NH(3) through the branchial epithelia. P. schlosseri probably developed such an 'environmental ammonia detoxification' capability because of its unique behavior of burrow building in the mudflats and living therein in a limited volume of water. In addition, the skin of P. schlosseri had low permeability to NH(3). Using an Ussing-type apparatus with 10 mmol l(-1) NH(4)Cl and a 1 unit pH gradient (pH 8.0 to 7.0), the skin supported only a very small flux of NH(3) (0.0095 micromol cm(-2) min(-1)). Cholesterol content (4.5 micromol g(-1)) in the skin was high, which suggests low membrane fluidity. Phosphatidylcholine, which has a stabilizing effect on membranes, constituted almost 50% of the skin phospholipids, with phosphatidyleserine and phsophatidylethanolamine contributing only 13% and 15%, respectively. More importantly, P. schlosseri increased the cholesterol level (to 5.5 micromol g(-1)) and altered the fatty acid composition (increased total saturated fatty acid content) in its skin lipid after exposure to ammonia (30 mmol l(-1) at pH 7.0) for 6 days. These changes might lead to an even lower permeability to NH(3) in the skin, and reduced back diffusion of the actively excreted NH(4)(+) as NH(3) or the net influx of exogenous NH(3), under such conditions.

Acids↗
PubMed2004

Dogmas and controversies in the handling of nitrogenous wastes: ureotely and ammonia tolerance in early life stages of the gulf toadfish, Opsanus beta.

The marine gulf toadfish (Opsanus beta) is an unusual teleost fish as it is able to switch between ammoniotelism and ureotelism in response to a variety of laboratory conditions. The present study integrates field work conducted in Biscayne and Florida Bays, USA with laboratory studies to examine ureotelism during the early life history stages of O. beta. Adult toadfish voluntarily nested in artificial shelters placed amongst seagrass beds and were found to be predominantly ureotelic under natural conditions as the internal shelter water had mean urea and ammonia concentrations (N=51) of 14.2+/-1.6 micro mol N l(-1) and 8.9+/-0.9 micro mol N l(-1), respectively. Toadfish successfully spawned in shelters, providing eggs, larvae and juvenile toadfish for laboratory study. In the lab, juvenile toadfish were also ureotelic and urea was excreted in pulsatile events that accounted for 62.0+/-5.9% of total urea-N excreted. Excretion rates of urea-N and ammonia-N were 1.018+/-0.084 micro mol N h(-1) g(-1) and 0.235+/-0.095 micro mol N h(-1) g(-1), respectively. Field-collected eggs, larvae and juveniles expressed significant levels of the ornithine-urea cycle enzymes carbamoyl-phosphate synthetase III, ornithine transcarbamylase and arginase and the accessory enzyme glutamine synthetase, all of which increased in activity as toadfish developed through early life stages. In juveniles, the ammonia 96-h LC(50) value was 875 micro mol N l(-1) and there was a 3-fold increase in ornithine transcarbamylase activity in the 1000 micro mol N l(-1) NH(4)Cl treatment. The results are discussed in the context of the causal factor(s) for ureotelism in toadfish. Furthermore, the results of this study suggest it is unlikely that the adaptive significance of ureotelism in toadfish is a means to prevent fouling nests with ammonia and in turn poisoning offspring; however, additional study is warranted.

Ammonia↗
PubMed2004

Dogmas and controversies in the handling of nitrogenous wastes: expression of arginase Type I and II genes in rainbow trout: influence of fasting on liver enzyme activity and mRNA levels in juveniles.

Through analysis of a cDNA library and third-party annotation of available database sequences, we characterized the full-length coding regions of rainbow trout (Oncorhynchus mykiss) Type I, Onmy-ARG01, and Type II, Onmy-ARG02, arginase genes. Two partial related arginase sequences, Onmy-ARG01b and Onmy-ARG02b, and a full-length zebrafish arginase coding region (Danio rerio), Dare-ARG02, are also reported. Comparison of vertebrate arginase sequences shows that both Type I and Type II genes in bony fishes contain a mitochondrial targeting N-terminal domain. This suggests that the cytosolic Type I arginase found in ureotelic vertebrates arose in the common ancestor of amphibia and mammals. Onmy-ARG01 and Onmy-ARG02 mRNA was detected in liver, kidney, gill, intestine, red muscle and heart tissues. Onmy-ARG01 was expressed at a significantly higher level relative to Onmy-ARG02 in liver and red muscle tissue. To investigate whether there was differential regulation of Onmy-ARG01 and Onmy-ARG02, juvenile trout were fasted for 6 weeks and hepatic enzyme activities and mRNA levels were compared with those of fed control fish. There was a 3-fold increase in liver arginase activity and a 2-fold increase in Onmy-ARG02 mRNA levels but no change in Onmy-ARG01 mRNA levels in fasted fish relative to fed fish. These findings indicate that both types of arginase genes are present and expressed in rainbow trout and that the pattern of expression varies between tissues. The increase in liver arginase activity after a 6-week fast is due, in part, to an increase in the expression of Onmy-ARG02 mRNA levels.

Amino Acid Sequence↗
PubMed2003

Evolution of glutamine synthetase in vertebrates: multiple glutamine synthetase genes expressed in rainbow trout (Oncorhynchus mykiss).

Glutamine synthetase (GSase) is a key enzyme in nitrogen metabolism and encoded by a single gene in mammals. Using PCR cloning techniques, including RT-PCR from total RNA and PCR from a cDNA library, we find evidence of four expressed GSase mRNAs for the tetraploid rainbow trout. For two of these mRNAs (Onmy-GS01, -GS02) we characterize the full-length coding regions, and for two others (Onmy-GS03, -GS04), we describe partial sequences. Northern analysis of Onmy-GS01, -GS02, -GS03 and -GS04 indicates that (1) Onmy-GS02 is expressed at higher levels relative to the other transcripts in most adult tissues, with the exception of brain and gill, where Onmy-GS01 is at the highest level, and (2) the tissue with the highest level of expression of all four transcripts is the brain, with decreasing levels in the intestine, liver, red muscle, gill/kidney, white muscle and heart. Clearly, rainbow trout possess multiple GSase genes with differing levels of tissue expression, implying manifold potential routes of regulation for this octameric enzyme. Our data also indicate that caution should be taken when interpreting mRNA expression data of a single gene, unless multiple genes have been ruled out. Consistent with a southern blot, phylogenetic and intron sequence analyses imply that the trout genes are encoded by at least four separate loci, belonging to two distinct evolutionary branches. Our data on rainbow trout, together with those from two full-length zebrafish Danio rerio GSase genes compiled from GenBank ESTs, support the idea that fish GSases are polyphyletic and that gene duplications have occurred at multiple points and in independent lineages throughout the evolution of bony fishes.

Amino Acid Sequence↗
PubMed2003

Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea.

Marine elasmobranchs retain urea and other osmolytes, e.g. trimethylamine oxide (TMAO), to counterbalance the osmotic pressure of seawater. We investigated whether a renal urea transporter(s) would be regulated in response to dilution of the external environment. A 779 bp cDNA for a putative skate kidney urea transporter (SkUT) was cloned, sequenced and found to display relatively high identity with facilitated urea transporters from other vertebrates. Northern analysis using SkUT as a probe revealed three signals in the kidney at 3.1, 2.8 and 1.6 kb. Upon exposure to 50% seawater, the levels of all three SkUT transcripts were significantly diminished in the kidney (by 1.8- to 3.5-fold). In response to environmental dilution, renal tissue osmolality and urea concentration decreased, whereas water content increased. There were no significant differences in osmolyte and mRNA levels between the dorsal-lateral bundle and ventral sections of the kidney. Taken together, these findings provide evidence that the downregulation of SkUT may play a key role in lowering tissue urea levels in response to external osmolality.

Amino Acid Sequence↗
PubMed2003

Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea.

Marine elasmobranch fishes maintain high urea concentrations and therefore must minimize urea loss to the environment in order to reduce the energetic costs of urea production. Previous studies have identified a facilitated urea transporter in the kidney of the dogfish. We examined mechanisms of urea transport in the kidney of the little skate Raja erinacea using an isolated brush-border membrane vesicle preparation. Urea uptake by brush-border membrane vesicles is by a phloretin-sensitive, non-saturable uniporter in the dorsal section and a phloretin-sensitive, sodium-linked urea transporter (Km = 0.70 mmol l(-1), Vmax = 1.18 micromol h(-1) mg(-1) protein) in the ventral section of the kidney. This provides evidence for two separate urea transporters in the dorsal versus ventral sections of the kidney. We propose that these two mechanisms of urea transport are critical for renal urea reabsorption in the little skate.

Adenosine Triphosphate↗