Tadpole Liver Research Articles

β-Glucuronidase in liver of tadpoles of Rana tigrina during growth and metamorphosis

1. 1. β-Glucuronidase activity of the liver showed a gradual increase in tadpole growth but during metamorphosis a twofold increase was observed. In adult growth the enzyme activity showed a significant decrease. 2. 2. β-Glucuronidase of the young tadpole liver showed a pH optimum at 4·8 but that from metamorphosing tadpoles showed pH optima at 4·2 and 4·8. 3. 3. The increased enzyme activity in metamorphosis was found to be located in the perivascular hepatic tissue. Such an increase seems to be related to the importation of β-glucuronidase-rich macrophages from the regressing tails.

Glutamate Dehydrogenase Biosynthesis in Amphibian Liver Preparations

Abstract Glutamate dehydrogenase (EC 1.4.1.3) (GDH) was studied with a system in vitro of tadpole and frog liver cube suspensions. A rise in GDH activity was observed in liver from tadpoles immersed in thyroxine (2.6 x 10-8 m) for 8 days. A small rise in enzyme activity was observed in vitro in liver cubes from premetamorphic tadpoles. In the presence of thyroxine (2.6 x 10-8 m to 2.6 x 10-10 m) or triiodothyropropionate (2.6 x 10-9 m or 2.6 x 10-11 m), the mean increase in the specific activity in vitro was twice that of the control over a 48-hour incubation period. Thyroxine stimulated synthesis de novo of immunoprecipitable glutamate dehydrogenase as well as soluble mitochondrial proteins. A specific stimulation of GDH synthesis relative to soluble mitochondrial protein synthesis in vitro was observed at a thyroxine concentration of 2.6 x 10-8 m. Higher concentrations of thyroxine resulted in a specific effect only in liver cubes prepared from tadpoles previously treated with thiourea. Synthesis de novo of GDH was inhibited by puromycin and actinomycin but the total enzyme activity in vitro was increased in the presence of these antibiotics. A continuous conversion of labeled nonimmunoprecipitable precursors into immunoprecipitable GDH was observed in vitro in the absence of any additives to the incubation system. In a single experiment, thyroxine itself appeared to stimulate precursor conversion. The half-disappearance time of labeled immunoprecipitable GDH was 21.2 ± 1.7 hours in vitro and 22.8 hours in vivo. Actinomycin and puromycin inhibited breakdown of immunoprecipitable GDH. Synthesis de novo, precursor conversion, and a rapid turnover of GDH were observed in vitro in liver cubes from premetamorphic and metamorphosing tadpoles as well as in the adult frog. The implications of these basic mechanisms of enzyme regulation are discussed.

The Effect of Thyroxine on Ribonucleic Acid Synthesis by Premetamorphic Tadpole Liver Cell Suspensions

Abstract Suspensions of tadpole liver cells have been developed as a model to further understanding of the biochemical differentiation of this tissue during thyroxine-induced metamorphosis. The cell population is homogeneous and stable; no cell division or DNA synthesis occurs in these isolated cells. RNA synthesis in cell suspensions is stimulated by thyroxine and triiodothyronine. The optimum concentration of both thyroxine and triiodothyronine for stimulation of RNA synthesis is about 10-7 m. Rapidly labeled RNA isolated from thyroxine-treated and nontreated cell suspensions sediments in the region between 4 and 10 S. The base composition of RNA synthesized by liver cells treated in vitro with thyroxine differs significantly from that of untreated liver cells and from bulk tadpole liver RNA. The ratio of cytosine plus guanine to total bases of RNA from thyroxine-treated cells is similar to that of DNA from this species. Neither thyroxine nor triiodothyronine had any effect on the incorporation of amino acids into protein. Frog serum stimulates RNA and protein synthesis. Rana catesbeiana serum causes the aggregation of isolated liver cells.

Mechanism of puromycin activation of tadpole liver glycogen synthetase

Tadpole liver glycogen synthetase (EC 2.4.1.11), which is completely glucose 6-phosphate dependent, is activated by insulin. Puromycin mimics the hormone and activates the enzyme to a similar degree. The antibiotic initiates a series of events which appears to result in the release of insulin. Inhibition of cyclic phosphodiesterase by puromycin raises hepatic cyclic AMP to a level which stimulates phosphorylase b kinase. The increased glycogenolysis, depletion of liver glycogen and hyperglycemia which result would lead to the release of insulin. By using alloxan-treated and pancreatectomized tadpoles we have shown that the puromycin activation of glycogen synthetase is contigent on functional β-cells in the pancrease to produce insulin.

Thyroxine stimulation of ornithine transcarbamylase activity and protein synthesis in tadpole (RNA catesbeiana) liver in organ culture

Ornithine transcarbamylase (OTC) activity and radioactive amino acid incorporation into protein decrease during the initial period that tadpole liver slices are maintained in organ culture; after 36 hours both activities increase. Thyroxine, added to the culture medium, stimulates OTC activity and the incorporation of amino acids. Light and electron microscopy observations are correlated with the biochemical alterations.

XV.—The metabolism of DNA in the liver during precocious induction of metamorphosis inRana catesbeiana

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers ofRana catesbeianatadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether thein vivochange was due to alterations in the pool sizes of the DNA precursors,in vitrostudies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

Primary mechanisms of thyroid hormone control of amphibian metamorphosis

Dramatic adaptive changes in numerous biochemical systems accompany the metamorphosis of the amphibian. These striking morphogenetic and molecular transformations are initiated and controlled by the thyroid hormones. That this differentiation process is providing a valuable experimental system for the study of the mechanism of action of the thyroid hormone is emphasized in a number of recent reviews. Current research progess has centered on the many alterations in RNA and protein metabolism in the developing tadpole liver, the induction of enzymes responsible for the advent of ureotelism, the process of tail resorption, and the remarkable effect of low temperatures on metamorphosis. Common mechanisms that appear to involve genetic control of protein and RNA biosynthesis have emerged to account for the changes observed in such diverse tissues as the liver and the tail. In this paper, we propose a sequence of molecular events during the first 2–3 hours after T 3 is administered to tadpoles by injection. During the first hour, T 3 promotes transport of nucleotides and amino acids into the liver. At the same time, there is a steadily increasing acceleration of protein and RNA biosynthesis that reaches its maximum at 60 min. Further interpretation of the data becomes more complicated because increased prehormonal RNA degradation and de novo nucleotide biosynthesis now impinge on the continuing macromolecular biosynthesis initiated earlier. The net result is the induction of new protein and RNA biosynthesis in the very early period after T 3 injection which eventually lead to the major synthetic events involving most of the intracellular structures of the liver.

Changes in the pathways for serine biosynthesis in the developing amphibian

1. 1. The pathways of serine biosynthesis utilizing either phosphorylated or nonphosphorylated intermediates, as well as the levels of activity of phosphoglycerate and d-glycerate dehydrogenases, have been investigated in tissues of frog at various stages of development. 2. 2. Serine biosynthesis occurs via the phosphorylated pathway in the unfertilized egg but by the nonphosphorylated route in liver of tadpoles undergoing metamorphosis and in adult liver. 3. 3. Phosphoglycerate dehydrogenase is present in unfertilized eggs and its activity remains relatively constant during the first the first 30 days following fertilization. Appreciable d-glycerate dehydrogenase activity is not detected until several days following hatching.

Effect of thyroxine treatment on the transfer of amino acids from aminoacyl transfer ribonucleic acid into protein by cell-free extracts from tadpole liver.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffect of thyroxine treatment on the transfer of amino acids from aminoacyl transfer ribonucleic acid into protein by cell-free extracts from tadpole liverBrian R. Unsworth and Philip P. CohenCite this: Biochemistry 1968, 7, 7, 2581–2588Publication Date (Print):July 1, 1968Publication History Published online1 May 2002Published inissue 1 July 1968https://doi.org/10.1021/bi00847a020RIGHTS & PERMISSIONSArticle Views26Altmetric-Citations13LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (727 KB) Get e-Alerts Get e-Alerts

Biochemical aspects of metamorphosis: changes in RNA associated with the transition from ammonotelism to ureotelism.

The relationship of RNA metabolism to the induction ot the enzyme carbamyl phosphate synthetase (the initial enzymatic step in biosynthesis of urea) in the liver of premetamorphic tadpoles exposed to thyroxine is reviewed. Treatment with thyroxine stimulates the synthesis of ribosomal, soluble, and “messenger” RNA fractions; the latter fraction shows the most rapid rate of synthesis in response to thyroxine. Properties of the “messenger” RNA fraction are reviewed. In keeping with the stimulation and formation of a “messenger” RNA fraction by thyroxine is the finding that thyroxine increases the efficiency of chromatin template for the synthesis of RNA. Although the present data stress the action of thyroxine on the efficiency of transcription, reference is made to the stimulatory effect of thyroxine at the level of translation in liver preparations from tadpoles. Thus, thyroxine has an effect at the levels of both transcription and translation.

Studies on Ribonucleic Acid Synthesis in Tadpole Liver during Metamorphosis Induced by Thyroxine: II. ANALYSIS OF RIBONUCLEIC ACID FRACTIONS

Abstract A method of preparing ribonucleic acid from tadpole liver has been described. It was found that tadpole liver RNA is unstable during the isolation procedure. Various factors, including temperature, the concentration of Mg++ in the homogenizing medium, and the presence of ribonuclease inhibitors, were found to be of importance in protecting RNA during its preparation. Three peaks with sedimentation coefficients of 29 S, 18 S, and 6 S were obtained by zonal sucrose density gradient centrifugation. The 6 S RNA was found in the purified ribosomes from mitochondrial and postmitochondrial fractions. The base ratios of 6 S RNA were different from those of the 18 S and 29 S fractions and the bulk RNA, showing in particular a low concentration of UMP. Some properties of RNase from tadpole liver have been described and discussed in relation to the base composition of 6 S RNA. The most rapidly labeled RNA appeared in the transfer RNA fraction when orotic-acid-14C was administered to untreated tadpoles. The RNA heavier than transfer RNA has the highest specific radioactivity 2 days after thyroxine treatment. The base composition of this RNA was analyzed with the use of 32Pi and found to be high in uridine 5'-phosphate (25 to 35%) in spite of the low value of the UMP in bulk RNA in this area. Rapidly labeled RNA which appears in the 6 to 10 S area has the properties of messenger RNA needed for induction of the enzymes in liver associated with metamorphosis.

Studies on Ribonucleic Acid Synthesis in Tadpole Liver during Metamorphosis Induced by Thyroxine: I. RELATION OF SYNTHESIS OF RIBONUCLEIC ACID AND OF CARBAMYL PHOSPHATE SYNTHETASE

Abstract Liver of tadpoles exposed to thyroxine shows a biphasic pattern of ribonucleic acid synthesis, with an initial sharp peak appearing after 36 to 48 hours. This is followed by a gradual increase during the period 6 to 15 days later. An induction of carbamyl phosphate synthetase was observed when tadpoles were treated with thyroxine for 2 days. The maximum specific radioactivity of RNA was seen in the microsomal RNA fraction 2 days after thyroxine treatment. Actinomycin D inhibited RNA synthesis in liver at all the stages of metamorphosis induced by thyroxine; however, delayed administration of actinomycin D did not inhibit induction of carbamyl phosphate synthetase. In the later stages of metamorphosis, temperature-dependent (thyroxine-independent) carbamyl phosphate synthetase synthesis was observed accompanied by a rapid RNA turnover.

Modification of tadpole liver chromatin by thyroxine treatment.

Modification of tadpole liver chromatin by thyroxine treatment.

The activity of lactate dehydrogenase isozymes during thyroxine-induced tadpole metamorphosis.

A new improved method to quantitate the amount of the isozymes of lactate dehydrogenase (LDH) has been devised. The method is sensitive and very reproducible. This method has been employed for studies on LDH isozymes during thyroxine-induced tadpole metamorphosis. Only one isozyme is present in tadpole liver whereas three isozymes are present in tadpole tail and brain. Thyroxine treatment produced a decrease of the total LDH activity of tadpole liver, tail, and brain; however, the relative amount of the isozyme F3in tail and brain is the only fraction which shows a decrease. The F1and F2fractions show an increase in relative amount. Thalidomide has no influence on the isozyme pattern of LDH in thyroxine-induced metamorphosis of the tadpole.

Comparative Study of Tadpole and Frog Glutamate Dehydrogenases

Abstract 1. There is approximately a 10-fold increase in the specific activity of liver glutamate dehydrogenase during the spontaneous metamorphosis of tadpoles (Rana catesbeiana). 2. Comparative studies of glutamate dehydrogenases from premetamorphic tadpole liver and adult frog liver revealed differences in kinetic constants for DPN and TPN, in the effect of temperature on initial rates with DPN as coenzyme, in the crude molecular weights estimated from sucrose density centrifugation, and in relative reactivities with l-amino acids other than glutamate. 3. An antibody produced in rabbits against crystalline frog liver glutamate dehydrogenase precipitates glutamate dehydrogenase preparations from frog, tadpole, beef, and chicken liver and affects their reaction kinetics in a similar manner.

Effect of carcinogenic and non-carcinogenic aminonitro compounds on the ultraviolet and blue fluorescence of tadpole liver cells

Model experiments with feeding tadpoles with carcinogenic and non-carcinogenic aminonitrocompounds revealed differences in the intensity of the ultraviolet and blue fluorescence of liver cells and in their fine morphology at early periods (15 min, 30 min, 1 h and 3 h) of contact with the tested substance. The authors suppose that these differences are due to the specific effect of a carcinogenic compound on mitochondria and cellular respiration.

ROLE OF CATALASES AND PEROXIDASES IN THE DEIODINATION OF THYROXINE IN FROG LIVER.

Previous data indicate that, in the frog, the transition from the larval to the adult stage is associated with a decrease or loss of the ability to deiodinate the thyroid hormones. Subsequent evidence suggests that the physiological deiodination of thyroxine (T4) is mediated by a hydrogen peroxide-peroxidase system in which tissue catalase acts as an inhibitor. In the present study, the role of catalases and peroxidases as determinants of the deiodinating activity of frog liver was assessed. Frog liver was shown to contain far more catalase activity than tadpole liver; considerable pyrogallol peroxidase activity was also observed. Comparative studies in the frog, rat, mouse and tadpole revealed that hepatic deiodinating activity varied inversely with catalase activity. Deiodinating activity was induced in homogenates of frog liver supplemented with ferrous ion or with an H2O2-generating system (glucose-glucose oxidase). 3-Amino-l,2,4-triazole, either administered in vivo or added in vitro, decreased catalase activity and induced significant T4- deiodinating activity in homogenates of frog liver. The stimulating effect of glucose oxidase was far greater in homogenates of liver from the amino-triazole treated frogs than from control animals. Azide added in vitro also decreased catalase activity and induced T4- deiodinating activity in homogenates of frog liver. Catalase activity was also greatly reduced by storing homogenates of frog liver for at least 24 hr. Significant deiodinating activity was found in these stored preparations. Dialysis had no greater effect than storage on the activity of hepatic catalase and T4- deiodinase. The results suggest that the H2O2 content of homogenates of frog liver is inadequate for the deiodination of T4. This is attributed to the relatively high concentration of hepatic catalases and to a limited generation of H2O2. (Endocrinology76: 479,1965)

Effect of corticosteroids on tadpole hepatic transaminases

Activities of aspartate-α-ketoglutarate (E.C.2.6.1.1), alanine-α-ketoglutarate (E.C.2.6.1.2) and tyrosine-α-ketoglutarate (E.C.2.6.1.5) transaminases in the livers of tadpoles were determined and the effect of corticoids and tyrosine upon the hepatic activities of these three transaminase studied. In contrast to mammalian livers, neither hydrocortisone nor tyrosine caused an appreciable increase in hepatic transaminase activities in tadpoles. The hepatic transaminase activities were not affected by treatment of the tadpoles with Metopirone, an inhibitor which blocks the biosynthesis of corticoids in vivo.

Composition and labeling patterns of “free” and protein amino acids in Rana catesbeiana tadpoles and frogs

The composition of free and protein amino acids, patterns of glucose-U-C 14 labeling, and amino acid pools of various tissues of Rana catesbeiana tadpoles and frogs were compared. It was demonstrated that there were no significant variations in the amino acid composition of whole organ proteins within one animal and of corresponding tissues of tadpole and frog. However, marked differences in several free amino acids in muscle were observed, with histidine being unusually abundant. Glucose-U-C 14 was incorporated into the protein amino acids alanine, glutamic acid, and aspartic acid in all tadpole tissues; in addition to these compounds, serine, glycine, and proline were also labeled in tadpole liver. Radioactivity levels of the free amino acids indicated that the synthesis of serine and glycine from glucose occurred more readily in frog liver than in tadpole liver. The time course of glucose-U-C 14 utilization for the formation of free and protein amino acids in tadpole liver was followed. It was observed that the maximum radioactivity in the free amino acids was reached before the peak of radioactivity was seen in the protein amino acids.

Studies on dehydrogenase activities during amphibian metamorphosis

The levels of activity of glutamic acid, lactic acid, glucose 6-phosphate, and malic acid dehydrogenases in prematamorphic tadpole liver and adult frog liver were determined. The level of glutamic acid dehydrogenase activity is six times higher in adult frog liver than in tadpole liver. Adult frog liver has slightly higher malic acid dehydrogenase, but lower glucose 6-phosphate and lactic acid dehydrogenase activities than tadpole liver. During thyroxine-induced metamorphosis (immersion of tadpoles in 2.6 · 10 −8 M thyroxine), the glutamic acid dehydrogenase activity of tadpole liver increased 150% after 20 days; lactic acid dehydrogenase and glucose 6-phosphate dehydrogenase activities decreased to 50–60% and to 75–80%, respectively, of the premetamorphic values. Tadpole and frog liver glutamic acid dehydrogenase is active with both DPN with the H 2O 2-generating system and 10 −6 M KI, for 2 or more minutes prior to addition of 131I, iodinating activity was destroyed (Table II). KI (10 −5 M) and tyrosine (10 −3 M) present during the incubation protected the enzyme (Table III).