The effects of long day length on liver antifreeze mRNA in the winter flounder, Pseudopleuronectes americanus

1984 ◽  
Vol 62 (8) ◽  
pp. 1456-1460 ◽  
Author(s):  
Ron M. Fourney ◽  
Garth L. Fletcher ◽  
Choy L. Hew
Keyword(s):  
Blot Hybridization ◽  
Winter Flounder ◽  
Day Length ◽  
Control Fish ◽  
Pituitary Hormone ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Pseudopleuronectes Americanus ◽  
Long Day ◽  
Afp Mrna

The effect of photoperiod on the seasonal accumulation of winter flounder (Pseudopleuronectes americanus) antifreeze polypeptide (AFP) mRNA in the liver was examined. AFP mRNA levels were identified and measured by cytoplasmic dot hybridization and Northern blot hybridization procedures utilizing a nick-translated antifreeze genomic clone. Flounder maintained under conditions of 15-h long day length have both a delayed appearance and decreased accumulation of AFP mRNA. December flounder maintained under long day length had the most significant decrease in AFP mRNA levels. It was estimated that these fish contained less than 0.6% of the AFP mRNA normally found in control fish. The seasonal fluctuation of AFP mRNA in both the experimental and control fish matched closely but preceded the rise and fall of plasma AFP levels. These results suggest that long day length suppresses the rate of transcription of antifreeze genes and supports the hypothesis that photoperiod may act as the initial cue for entraining the precise activation of AFP synthesis. A pituitary hormone may be the mediator.

Comparative effects of food restriction, fasting, diabetes and thyroidectomy on growth hormone and thyrotropin gene expression in the rat pituitary

1995 ◽  
Vol 133 (1) ◽  
pp. 110-116 ◽  
Author(s):  
Manuela Rodriguez ◽  
Felipe Rodriguez ◽  
Trinidad Jolin ◽  
Pilar Santisteban
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Food Restriction ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Restricted Feeding ◽  
Rat Pituitary ◽  
Ad Libitum ◽  
Ad Libitum Intake

Rodriguez M, Rodriguez F, Jolin T, Santisteban P. Comparative effects of food restriction, fasting, diabetes and thyroidectomy on growth hormone and thyrotropin gene expression in the rat pituitary. Eur J Endocrinol 1995;133:110–6. ISSN 0804–4643 To examine the molecular basis for the decreased pituitary growth hormone (GH) and thyrotropin (TSH) content during restricted feeding, fasting and diabetes, we measured steady-state levels of mRNA for TSH-α TSH-β and GH in the pituitary from normal rats either fed ad libitum (C), limited to 75%, 50% and 25% (FR75, FR50, FR25, respectively) of ad libitum intake, or deprived of food for 2 and 4 days (F2 and F4, respectively), and also in streptozotocin-diabetic (D) and D insulin-treated animals. The results from these experimental groups were compared with those in thyroidectomized (Tx) rats. Pituitary mRNA was quantified by Northern blot hybridization with cDNA probes specific for rat TSH-α, TSH-β and GH. Although changes in the pituitary GH mRNA during restricted feeding, fasting and diabetes were similar qualitatively to those induced by hypothyroidism, GH mRNA levels in Tx rats (> 10% of C values) were less than in the other experimental groups (p < 0.001). Pituitaries from FR50, FR25 and D rats also contained less GH mRNA than F2 and F4 animals (p < 0.05). Thyroidectomy resulted in a marked increase in both TSH-β and TSH-α mRNAs, the changes in TSH-β mRNA being greater than those in TSH-α mRNA. In contrast, FR50, FR2 5, F2, F4 and D rats exhibited a decrease in pituitary TSH-β mRNA (60%, 50%, 35%, 36% and 33%, respectively, of C values; p < 0.01–0.05) and in TSH-α mRNA levels (81%, 64%, 46%, 43% and 36%, respectively, of normal values; p < 0.02–0.05), TSH-β mRNA showing the greater changes. However, pituitaries from F2, F4 and D rats contained less TSH-β and TSH-α mRNA levels than FR50 and FR25 animals (p < 0.05). Insulin therapy partially restored the changes in mRNA for GH, TSH-β and TSH-α observed in D rats. In addition, the pituitary nuclear triiodothyronine in Tx, FR50, FR25, F2, F4 and D rats was reduced to 19%, 73%, 52%, 76%, 51% and 41%, respectively of C values (p < 0.05–0.001). These data suggest that GH, TSH-α and TSH-β gene expression are modulated by metabolic and/or endocrine changes accompanying restricted feeding, fasting and diabetes. P Santisteban, Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, Spain

Effect of hypothyroidism and thyroid hormone treatment of the rat on hepatic Spot 14 and thyroxine binding prealbumin mRNAs

1989 ◽  
Vol 121 (3) ◽  
pp. 383-388 ◽  
Author(s):  
J. A. Franklyn ◽  
S. King ◽  
J. A. Ahlquist ◽  
M. C. Sheppard
Keyword(s):  
Thyroid Hormone ◽  
Hormone Replacement ◽  
Dose Range ◽  
Blot Hybridization ◽  
Ventricular Myocardium ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Thyroid Status ◽  
Spot 14 ◽  
Stimulation Of

Abstract. We have examined the influence of hypothyroidism and thyroid hormone replacement on hepatic levels of Spot 14 and thyroxine binding prealbumin mRNAs determined by dot hybridization and by Northern blot hybridization to specific complementary DNA probes. A marked reduction in Spot 14 mRNA was demonstrated in hypothyroidism, compared with the euthyroid state. T3 replacement of hypothyroid rats, using a wide dose range of T3 (1–20 μg) and 6 and 72 h time points, demonstrated no sustained effect at 6 h, but a dose-dependent stimulation of Spot 14 mRNA at 72 h after daily treatment with T3 was commenced. In contrast, no effect of hypothyroidism or T3 replacement on hepatic levels of thyoxine binding prealbumin mRNA was demonstrated, indicating the specificity of thyroid hormone action. T3 treatment of euthyroid rats was also associated with a dose-related stimulation of Spot 14 mRNA levels. The effect of hypothyroidism and T3 treatment of the rat on hepatic Spot 14 mRNA contrasts with divergent regulatory influences of thyroid status in the anterior pituitary and ventricular myocardium demonstrated using identical animal models, indicating the tissue specific influences of thyroid status.

Sepsis-induced attenuation of glucagon and 8-BrcAMP modulation of the phosphoenolpyruvate carboxykinase gene

1995 ◽  
Vol 269 (3) ◽  
pp. R584-R591 ◽  
Author(s):  
C. S. Deutschman ◽  
A. De Maio ◽  
M. G. Clemens
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Blot Hybridization ◽  
Cecal Ligation ◽  
Acute Phase Reactant ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Hormonal Milieu ◽  
Krebs Buffer

Sepsis is associated with alterations in hepatic gluconeogenesis. We have previously demonstrated that this change is associated with reduced expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene, despite an endogenous hormonal milieu that should favor increased expression of the gene. To further elucidate the mechanisms involved, we induced sepsis in fasted Sprague-Dawley rats via cecal ligation and single puncture, with sham-operated animals serving as controls, and we performed two sets of experiments. First, liver tissue was obtained from septic and sham-operated animals at 2, 6, 16, and 24 h after the induction of sepsis. Northern blot hybridization analysis revealed a progressive, sepsis-induced decrease in expression of PEPCK and an increase in the expression of beta-fibrinogen, an acute-phase reactant. In the second set of experiments, we tested whether this reduced expression resulted from an attenuated response to 1) glucagon and 2) 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Twenty-four hours after the induction of sepsis, the liver was isolated and perfused with either Krebs buffer with substrate only (unstimulated controls), Krebs buffer + substrate + 10(-8) M glucagon, or Krebs buffer + substrate + 10(-5) M 8-BrcAMP. In sham-operated animals, perfusion with glucagon increased PEPCK mRNA levels and activity, whereas perfusion with buffer alone did not change mRNA levels and decreased activity. Glucagon perfusion of septic livers did not change either PEPCK mRNA levels or activity. Perfusion of sham-operated animals with 8-BrcAMP increased PEPCK mRNA levels and activity, whereas perfusion with buffer alone resulted in a decrease in mRNA levels and activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of liver angiotensinogen and kidney renin mRNA levels by angiotensin II

1990 ◽  
Vol 258 (1) ◽  
pp. E1-E6 ◽  
Author(s):  
A. Nakamura ◽  
H. Iwao ◽  
K. Fukui ◽  
S. Kimura ◽  
T. Tamaki ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Mrna Level ◽  
Control Level ◽  
Renin Angiotensin System ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Ang Ii ◽  
Low Sodium ◽  
Renin Mrna

The current study was designed to evaluate the role of angiotesin II (ANG II) in the regulation of renin mRNA and angiotensinogen mRNA levels. We investigated the changes in renin mRNA levels in the kidney and angiotensinogen mRNA levels in the liver induced by ANG II infusion and by inhibition of the endogenous renin-angiotensin system with enalapril or saralasin in rats. mRNAs for angiotensinogen and renin were measured by densitometric analysis of Northern blot hybridization. After a 4-h intravenous infusion of ANG II (0.2 micrograms.kg-1.min-1), angiotensinogen mRNA level in the liver increased 2.5-fold to the control level, and renin mRNA level in the kidney decreased by 45%. Four hours after treatment with a single dose of enalapril (3 mg/kg po), angiotensinogen mRNA level in the liver decreased by 50%, and renin mRNA level in the kidney increased 1.5-fold to the control level. These mRNA levels returned to control levels 8 h after treatment. After a 4-h infusion of saralasin (2.5 micrograms.kg-1.min-1) in the low-sodium state, angiotensinogen mRNA level in the liver decreased by 45%, and renin mRNA level in the kidney increased twofold to the control level. These findings suggest that ANG II is one of the factors that regulate angiotensinogen mRNA level in the liver and renin mRNA level in the kidney.

Evidence for Antifreeze Protein Gene Transfer in Atlantic Salmon (Salmo salar)

1988 ◽  
Vol 45 (2) ◽  
pp. 352-357 ◽  
Author(s):  
Garth L. Fletcher ◽  
Margaret A. Shears ◽  
Madonna J. King ◽  
Peter L. Davies ◽  
Choy L. Hew
Keyword(s):  
Atlantic Salmon ◽  
Salmo Salar ◽  
Cold Water ◽  
Restriction Enzymes ◽  
Antifreeze Protein ◽  
Winter Flounder ◽  
Control Fish ◽  
Pseudopleuronectes Americanus ◽  
Atlantic Salmon Salmo Salar ◽  
Antifreeze Polypeptides

Atlantic salmon (Salmo salar) freeze to death if they come into contact with ice at water temperatures below −0.7 °C. Consequently, sea-pen culture of this species in cold water is severely limited. Winter flounder (Pseudopleuronectes americanus) survive in ice-laden seawater by producing a set of antifreeze polypeptides (AFP). We are attempting to make the Atlantic salmon more freeze resistant by transferring antifreeze protein genes from the winter flounder to the genome of the salmon. Salmon eggs were microinjected with linearized DNA after fertilization. Individual fingerlings (1–2 g) were analyzed for flounder AFP genes by genomic Southern blotting. DNA from 2 out of 30 fingerlings showed hybridization to the flounder DNA probe. Hybridization bands following cleavage by restriction enzymes Sst l and Bam HI were identical to those of the injected DNA. Hybridization following Hind III digestion indicated that the flounder AFP gene was linked to the salmon genome. These hybridization signals were absent in the DNA from control fish. The intensity of the hybridization signals indicated that there was on average at least one copy of the AFP gene present per cell.

Pro-opiomelanocortin messenger RNA levels increase in the fetal sheep pituitary during late gestation

1991 ◽  
Vol 131 (3) ◽  
pp. 483-489 ◽  
Author(s):  
K. Yang ◽  
J. R. G. Challis ◽  
V. K. M. Han ◽  
G. L. Hammond
Keyword(s):  
Messenger Rna ◽  
Blot Hybridization ◽  
Late Gestation ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Plasma Acth ◽  
Mrna Accumulation ◽  
Fetal Sheep ◽  
Fetal Plasma ◽  
Pomc Mrna

ABSTRACT Plasma levels of ACTH and cortisol in fetal sheep increase progressively during late pregnancy, providing the stimulus for birth. However, little information is available concerning either sources of pro-opiomelanocortin (POMC, the precursor to ACTH) or changes in POMC gene expression, which may be responsible for the elevated fetal plasma ACTH concentrations. We therefore studied the relative amount of POMC mRNA in fetal sheep hypothalami, anterior pituitaries and adrenals at discrete times of pregnancy between day 60 and term (approximately 145 days) and from newborn lambs. Total RNA from these tissues was analysed by Northern blot hybridization using a human POMC DNA probe, and the amount of POMC mRNA was expressed relative to the signal obtained for 18S ribosomal RNA. A single 1·2 kb transcript was detected by day 60 in the anterior pituitary, and its relative amount did not change significantly until after days 125–130. Pituitary POMC mRNA levels increased significantly at days 138–143, remained elevated at term and increased further in newborn lambs. In contrast, POMC mRNA was undetectable in hypothalami and adrenal glands of fetuses at all ages. The results suggested that the prepartum rise in plasma ACTH concentrations in fetal sheep is due to increased POMC biosynthesis in the fetal pituitary. The increase in POMC mRNA occurs at a time when fetal plasma cortisol concentrations are elevated, indicating that the negative feedback effects of circulating glucocorticoids on the fetal hypothalamicpituitary axis may be obscured by other mechanisms that increase pituitary POMC mRNA accumulation during the last week of gestation. Journal of Endocrinology (1991) 131, 483–489

Tissue distribution of fish antifreeze protein mRNAs

1992 ◽  
Vol 70 (4) ◽  
pp. 810-814 ◽  
Author(s):  
Zhiyuan Gong ◽  
Garth L. Fletcher ◽  
Choy L. Hew
Keyword(s):  
Northern Blot Analysis ◽  
Antifreeze Protein ◽  
The Body ◽  
Winter Flounder ◽  
Type I ◽  
Pseudopleuronectes Americanus ◽  
Sea Raven ◽  
Hemitripterus Americanus ◽  
Afp Mrna ◽  
Distribution Of Fish

The presence of fish antifreeze protein (AFP) mRNA was examined in a variety of tissues from the winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), and ocean pout (Macrozoarces americanus), each of which contains one of the three known AFP types. Northern blot analysis indicates that whereas the AFP mRNA is restricted to liver in sea raven (type II AFP), significant amounts of mRNA are present in many other tissues in both winter flounder (type I) and ocean pout (type III). These results indicate that in sea raven, antifreeze protein synthesis only occurs in the liver, whereas in the ocean pout and winter flounder, synthesis occurs in many tissues throughout the body. These investigations are relevant to understanding the mode of action of these polypeptides.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

1989 ◽  
Vol 67 (10) ◽  
pp. 2520-2527 ◽  
Author(s):  
King Ming Chan ◽  
William S. Davidson ◽  
Choy L. Hew ◽  
Garth L. Fletcher
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Cadmium Chloride ◽  
Gill Filament ◽  
Winter Flounder ◽  
Mrna Levels ◽  
Pseudopleuronectes Americanus ◽  
Coding Region ◽  
Liver And Kidney ◽  
Time Required

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

The effects of hypophysectomy on seasonal changes in plasma freezing-point depression, protein 'antifreeze,' and Na+ and Cl− concentrations of winter flounder (Pseudopleuronectes americanus)

1978 ◽  
Vol 56 (1) ◽  
pp. 109-113 ◽  
Author(s):  
G. L. Fletcher ◽  
C. M. Campbell ◽  
C. L. Hew
Keyword(s):  
Seasonal Changes ◽  
Freezing Point ◽  
Early Summer ◽  
Winter Flounder ◽  
Day Length ◽  
Freezing Point Depression ◽  
Pseudopleuronectes Americanus ◽  
Ambient Seawater ◽  
Annual Increase ◽  
Annual Changes

The annual changes in plasma Na+ and Cl− concentrations took place in the absence of the pituitary, although the magnitude of the change was significantly reduced. The annual increase in plasma freezing-point depression also occurred in the absence of the pituitary. However the decrease normally observed in the spring and early summer did not occur.Sham-operated winter flounder transferred from ambient seawater (−1 °C) and day length to warm water (6–12 °C) and 18-h day length showed a reduction in plasma Cl− concentration and freezing-point depression and a loss of the protein 'antifreeze.' Hypophysectomized flounder treated in the same way showed a reduction in plasma Cl−, but no decline in freezing-point depression and protein 'antifreeze.'These results suggest that an intact pituitary is necessary for the disappearance of the protein 'antifreeze' from the plasma of the winter flounder.

Circannual cycles of blood plasma freezing point and Na+ and Cl− concentrations in Newfoundland winter flounder (Pseudopleuronectes americanus): correlation with water temperature and photoperiod

1977 ◽  
Vol 55 (5) ◽  
pp. 789-795 ◽  
Author(s):  
G. L. Fletcher
Keyword(s):  
Freezing Point ◽  
Sampling Site ◽  
Winter Flounder ◽  
Day Length ◽  
Freezing Point Depression ◽  
Pseudopleuronectes Americanus ◽  
Annual Cycles ◽  
Sea Floor ◽  
Water And Sediment ◽  
Circannual Cycles

A 4-year field program was conducted on the winter flounder to correlate changes in plasma Na+, Cl−, and freezing-point depression with sea-floor water and sediment temperatures, day length, salinity, depth of capture, and observations on burrowing in sediments.Plasma Na+, Cl−, and freezing-point depression showed annual cycles with maxima in winter (January–April) (temperature −1.1 to −1.4 °C) and minima in summer (July–October) (10–14 °C). The change in freezing-point depression from summer to winter was about 0.65 °C, 20% of this was attributable to Na+ and Cl− and the remaining 80% to the presence of an 'antifreeze.' The data suggest that plasma 'antifreeze' appeared in November (4–6 °C) and disappeared during May (−1.0 to 3 °C). During the winter the flounder were found only in the deeper areas of the sampling site and were usually buried up to 12–15 cm in the sediments which were warmer (0.1 to 0.4 °C) than the seawater.The plasma Na+, Cl−, and freezing-point depression of winter flounder held in the laboratory for 7 days were always significantly lower than the field-sampled fish. The differences between these two groups was greatest during the summer, suggesting that the effects of 'stress' during capture differ seasonaly.

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