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V A Galton

Publications and source records attributed to V A Galton.

At least 19 recordsLinked to original sources
PubMed2001

The type 2 iodothyronine deiodinase is expressed in the rat uterus and induced during pregnancy.

Thyroid hormones are of considerable importance for vertebrate reproductive function and during development. To further assess the role of these compounds in this capacity, we examined the expression pattern of the type 2 iodothyronine deiodinase (D2), which converts T(4) to the more active hormone T(3), in the rat uterus in both the nonpregnant and the pregnant state. D2 activity was identified as the predominant, if not only, 5'-deiodinase in the nonpregnant rat uterus. The expression of D2 messenger RNA was located by in situ hybridization to the endometrial stromal cells, where the signal was particularly enriched in the region adjacent to the epithelial cells of the uterine lumen. During pregnancy, D2 activity increased, peaking on day 17 of gestation (embryonic day 17). At that time, uterine D2 activity exceeded that in the placenta, as well as that in the fetal tissues. In the earlier stages of pregnancy before placental formation (e.g. embryonic days 10-11), D2 messenger RNA in the rat uterus was located outside the decidual tissue, which was observed, as in previous studies, to highly express the inactivating type 3 deiodinase. In summary, the rat uterus, particularly during pregnancy, seems to be a site of active thyroid hormone metabolism, presumably designed to maintain the optimal thyroid hormone environment for both the fetus and the maternal uterine tissue.

Animals↗
PubMed2001

Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4.

The type 2 deiodinase (D2), a selenoenzyme that catalyzes the conversion of T4 to T3 via 5'-deiodination, is expressed in the pituitary, brain, brown adipose tissue (BAT), and the reproductive tract. To examine the physiological role of this enzyme, a mouse strain lacking D2 activity was developed using homologous recombination. The targeting vector contained the Neo gene in place of a 2.6-kb segment of the Dio2 gene. This segment comprises 72% of the coding region and includes the TGA codon that codes for the selenocysteine located at the active site of the enzyme. Mice homologous for the targeted deletion [D2 knockout (D2KO)] had no gross phenotypic abnormalities, and development and reproductive function appeared normal, except for mild growth retardation (9%) in males. No D2 activity was observed in any tissue in D2KO mice under basal conditions, or under those that normally induce this enzyme such as cold-exposure (BAT) or hypothyroidism (brain, BAT, and pituitary gland). Furthermore, no D2 activity was present in cultured astrocytes, nor could it be induced by treatment of the cells with forskolin. Although D2 mRNA transcripts were detected in BAT RNA obtained from cold-exposed wild-type (WT) mice, none was detected in BAT RNA from comparably-treated D2KO mice. Levels of D1 in the liver, thyroid, and pituitary were the same in WT and D2KO animals, whereas D3 activity in D2KO cerebrum was twice that in WT cerebrum. Serum T3 levels were comparable in adult WT and D2KO mice. However, serum T4 and TSH levels were both elevated significantly (40% and 100%, respectively) in the D2KO mice, suggesting that the pituitary gland of the D2KO mouse is resistant to the feedback effect of plasma T4. This view was substantiated by the finding that serum TSH levels in hypothyroid WT mice were suppressed by administration of either T4 or T3, but only T3 was effective in the D2KO mouse. The data also suggest that the clearance of T4 from plasma was reduced in the D2KO mouse. In summary, targeted inactivation of the selenodeiodinase Dio2 gene results in the complete loss of D2 activity in all tissues examined. The increased serum levels of T4 and TSH observed in D2KO animals demonstrate that the D2 is of critical importance in the feedback regulation of TSH secretion.

Adipose Tissue, Brown↗
PubMed2000

Iodothyronine sulfotransferase activity in rat uterus during gestation.

In developing mammals, we and others demonstrated that sulfation is an important pathway in the metabolism of thyroid hormone, and there is significant fetal-maternal transfer of sulfated iodothyronine. In the present study, we characterized a novel iodothyronine sulfotransferase (IST) in pregnant rat uterus. (125)I-labeled 3,3'-diiodothyronine (T(2)), T(3), rT(3), and T(4) were used as substrates with unlabeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate donor. Sulfated iodothyronine products were separated by Sephadex LH-20 column and further identified on reverse phase HPLC. We measured IST activity in pregnant rat uterus by incubating 1 microM substrate, 50 microM PAPS, and 50 microg cytosol protein, pH 7.2, 30 min at 37 degrees C. The results show that the substrate preference of the uterine IST activity is: T(2 )> rT(3 )> T(3)> T(4); the pH optimum is 6.0 for T(2). The K(m) and V:(max) (for gestational day 21 uterus) for T(2) are 0.62 microM and 3466 pmol/mg protein/h, respectively; for PAPS the values are 2.6 microM and 1523 pmol/mg protein/h, respectively. During pregnancy, the total activities exhibit a U-shaped curve with minimum activity at day 13 of gestation; while a thermostable activity increases significantly near term. In summary, there is significant uterine IST that varies during pregnancy. The role of this uterine sulfotransferase activities in regulating the bioavailability of thyroid hormone in the developing fetus remains to be elucidated.

Animals↗
PubMed2000

Effects of selenium deficiency on tissue selenium content, deiodinase activity, and thyroid hormone economy in the rat during development.

The iodothyronine deiodinases, D1, D2, and D3, all contain selenium (Se) in the form of selenocysteine at their active sites, and they play crucial roles in determining the circulating and intracellular levels of the active thyroid hormone (TH), T3. However, not only are serum T3 levels normal in Se-deficient rats but phenotypic and reproductive abnormalities are minimal, and it has been suggested that regulatory mechanisms exist to conserve Se in critical tissues. The present study was designed to determine, in rats: 1) whether the effects of Se-deficiency are greater in the fetus and neonate than in the adult; 2) whether there are tissues other than brain and thyroid in which deiodinase activities are maintained; 3) whether the maintenance of deiodinase activity in a specific tissue is associated with a concomitant preservation of Se level in that tissue; and 4) whether TH economy and general health is maintained over several generations. The tissues studied included liver, cerebrum, thyroid, pituitary, skin, brown adipose tissue, uterus, ovary, testis, placenta, and the implantation site (uterus plus contents) at E9. The results have revealed that, with the exception of liver, skin, and nonpregnant uterus, all of the tissues studied maintained substantial deiodinase activity (>50%) during prolonged Se-deficiency. Second, although the ability of a tissue to maintain deiodinase activity in the face of dietary Se deprivation was associated in some tissues with a concomitant local preservation of Se concentration, this was not the case for all tissues. Only when Se levels were decreased by more than 80% was deiodinase activity markedly decreased. Third, the effects of Se-deficiency were no greater in the fetus than in the adult; and fourth, at the level of Se-deficiency employed in this study, TH economy and general health were successfully maintained over six generations of Se-deficient rats. How Se levels are maintained in specific tissues, whether Se is sequestered in specific cells of a tissue or organ during dietary Se deprivation, and the precise mechanisms by which plasma T3 levels are maintained in Se-deficient animals remain unanswered. Further insights may be gained by using diets that are even lower in Se than those that were used herein and/or by conducting studies using radioactive forms of Se and thyroid hormones.

Aging↗
PubMed1999

Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase.

Although thyroid hormones are critically important for the coordination of morphogenic processes in the fetus and neonate, premature exposure of the embryo to levels of the hormones present in the adult is detrimental and can result in growth retardation, malformations, and even death. We report here that the pregnant rat uterus expresses extremely high levels of the type 3 iodothyronine deiodinase (D3), which inactivates thyroxine and 3,3', 5-triiodothyronine by 5-deiodination. Both D3 mRNA and activity were present at the implantation site as early as gestational day 9 (E9), when expression was localized using in situ hybridization to uterine mesometrial and antimesometrial decidual tissue. At later stages of gestation, uterine D3 activity remained very high, and the levels exceeded those observed in the placenta and in fetal tissues. After days E12 and E13, as decidual tissues regressed, D3 expression became localized to the epithelial cells lining the recanalized uterine lumen that surrounds the fetal cavity. These findings strongly suggest that the pregnant uterus, in addition to the placenta, plays a critical role in determining the level of exposure of the fetus to maternal thyroid hormones.

Animals↗
PubMed1999

Expression profiles of the three iodothyronine deiodinases, D1, D2, and D3, in the developing rat.

Thyroid hormone (TH) is essential for normal development in vertebrate species. Although the mechanisms by which TH regulates developmental processes are not fully understood, intracellular T3 levels are likely to be a critical aspect of the process. Furthermore, as different tissues and organs have specific temporal patterns of development, their T3 requirements may vary widely. Differential regulation of intracellular T3 levels in peripheral tissues as a result of differences in the activities of the three iodothyronine deiodinases (D1, D2, and D3) could offer an important means of achieving coordination of T3-dependent developmental processes among tissues. To obtain evidence for this concept we have documented the levels of expression of all three types of deiodinase in 11 tissues of the fetus, the neonate, and the adult rat. In most fetal tissues, D3 was the predominant deiodinase, but it declined after birth as the activities of D1 and D2 increased. Exceptions to this pattern were skin and brown adipose tissue (BAT), in which D2 activity was highest in the fetus, and testis and thyroid in which D2 activity was higher in the neonate than in the adult. D1 was the only 5'D enzyme expressed in liver, kidney and intestine at all stages studied, and D3 was not expressed in these tissues after birth. Thyroid, pituitary, and BAT expressed either D2 or D2 plus D1, but did not express D3 at any stage studied. Cerebrum, cerebellum, ovary, testis, skin, and placenta expressed all three deiodinases. Two other points were evident. First, the maximum 5'D activity attained, and thus presumably the amount of T3 generated, in liver, kidney, intestine, thyroid, pituitary, and BAT was very much higher than that in cerebrum, cerebellum, ovary, testis, skin, and placenta. Second, in the tissues where 5'D activity was relatively low, coexpression of D3 with D1 and D2 was the general rule, suggesting the need for very tight control of intracellular T3 levels. The findings are consistent with the view that the deiodinases play a major role in achieving the intracellular T3 levels that are optimal for the development of each tissue. Additional studies are in progress to demonstrate the functional consequences of these deiodinase expression patterns.

Aging↗
PubMed1999

Cloning of a 5.8 kb cDNA for a mouse type 2 deiodinase.

From studies with their cDNAs, the types 1 and 3 deiodinases (D1 and D3) have been shown unequivocally to be selenoproteins. Studies with recently cloned cDNAs for the mammalian type 2 deiodinase (D2) indicate that they also code for selenoproteins. However, these D2 cDNAs are not full length and they do not contain an essential selenocysteine insertion sequence (SECIS) in their 3'UTR; a heterologous SECIS had to be ligated to the coding region before expression of the D2 could be achieved. Thus their role as cDNAs for the native D2 is open to question. We now report the cloning of a 5.8 kb cDNA for the mouse D2. This cDNA contains a SECIS in its 3'UTR located more than 4.5 kb from the coding region. When the mRNA transcribed in vitro from this cDNA is injected into X. laevis oocytes, a deiodinase with characteristics of D2 is expressed.

3' Untranslated Regions↗
PubMed1997

The deiodinase family of selenoproteins.

The realization some forty years ago that several iodothyronine compounds are present in the circulation suggested that deiodination occurs in various tissues. Subsequently, deiodination was indeed documented in in vivo studies. Later, using in vitro assay techniques, three deiodinase processes, termed types 1, 2 and 3, were defined that differed in terms of tissue distribution, reaction kinetics, efficiency of substrate utilization and sensitivity to inhibitors. Although purification of the deiodinase enzymes has continued to be problematic, recent molecular cloning studies have identified cDNAs for these three deiodinase isoforms from multiple species. These cDNAs have provided important insights into the structural characteristics of this family of enzymes. Foremost among the structural features has been the demonstration that all three deiodinase isoforms contain at their active site the uncommon amino acid selenocysteine which is of critical importance to their catalytic activity. The availability of cDNAs for these enzymes provides important reagents for pursuing additional studies aimed at defining their biochemical features and roles in thyroid hormone economy.

Animals↗
PubMed1997

The type 2 and type 3 iodothyronine deiodinases play important roles in coordinating development in Rana catesbeiana tadpoles.

In developing Rana catesbeiana tadpoles, the timing of the thyroid hormone (TH)-dependent metamorphic responses varies markedly among tissues. Yet at any one time these tissues are exposed to the same plasma concentration of TH, suggesting that TH action is regulated in part at the level of the peripheral tissues. A major factor in TH action is the intracellular level of the active TH, T3. This level is dependent not only on the plasma concentration of TH (mostly T4) but also on the intracellular activities of the type 2 5'-deiodinase (D2) and the type 3 5-deiodinase (D3), which are responsible, respectively, for generating and degrading T3. (D1 is not present in this species.) To determine whether differential expression of D2 and D3 among tissues could be a significant factor in the coordination of metamorphic events, the ontogenic profiles of the two enzyme activities and corresponding messenger RNA levels in most tissues of R. catesbeiana tadpoles have been documented. The profiles of D2 expression in tail, hindlimb, forelimb, intestine, skin, and eye differed markedly at both activity and messenger RNA levels, but it was notable that expression was invariably highest in a given tissue at the time of its major metamorphic change. D2 expression was very low in brain and heart and did not vary during development. D2 was not expressed in liver, kidney, or red blood cells. With the exception of red blood cells, D3 expression was detected in all tissues studied. Furthermore, it was evident that in tissues that expressed both deiodinase genes, the two expression profiles were comparable, indicating a potential for tight control of intracellular T3 levels. Direct evidence of the importance of the intracellular conversion of T4 to T3 for TH-dependent metamorphic events was obtained in tadpoles in which endogenous TH synthesis was blocked with methimazole, and the activities of D2 and D3 were inhibited by iopanoic acid. This treatment inhibited metamorphosis. The inhibition could be overcome by the concomitant administration of replacement levels of T3, but not T4. These results strongly support the view that coordinated development in amphibia depends in part on the tissue-specific expression patterns of the D2 and D3 genes, which ensure that the requisite level of intracellular T3 is attained in a given tissue, regardless of the current level of circulating TH, at the appropriate stage of metamorphosis.

Animals↗
PubMed1996

Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues.

The deiodination of thyroid hormones in extrathyroidal tissues plays an important role in modulating thyroid hormone action. The type II deiodinase (DII) converts thyroxine to the active hormone 3,5,3'-triiodothyronine, and in the rat is expressed in the brain, pituitary gland, and brown adipose tissue (BAT). Complementary DNAs (cDNAs) for the types I and III deiodinases (DI and DIII, respectively) have been isolated and shown to code for selenoproteins. However, information concerning the structure of the mammalian DII remains limited, and the pattern of its expression in human tissues is undefined. We report herein the identification and characterization of rat and human DII cDNAs. Both code for selenoproteins and exhibit limited regions of homology with the DI and DIII. In the rat pituitary and BAT, DII mRNA levels are altered more than 10-fold by changes in the thyroid hormone status of the animal. Northern analysis of RNA derived from human tissues reveals expression of DII transcripts in heart, skeletal muscle, placenta, fetal brain, and several regions of the adult brain. These studies demonstrate that: (a) the rat and human DII are selenoproteins, (b) DII expression in the rat is regulated, at least in part, at the pretranslational level in some tissues, and (c) DII is likely to be of considerable physiologic importance in thyroid hormone economy in the human fetus and adult.

Adipose Tissue, Brown↗
PubMed1995

Cloning of a cDNA for the type II iodothyronine deiodinase.

Three types of iodothyronine deiodinase have been identified in vertebrate tissues. cDNAs for the types I and III have been cloned and shown to contain an inframe TGA that codes for selenocysteine at the active site of the enzyme. We now report the cloning of a cDNA for a type II deiodinase using a reverse transcription/polymerase chain reaction strategy and RNA obtained from Rana catesbeiana tissues. This cDNA (RC5'DII) manifests limited but significant homology with other deiodinase cDNAs and contains a conserved in-frame TGA codon. Injection of capped in vitro synthesized transcripts of the cDNA into Xenopus laevis oocytes results in the induction of deiodinase activity with characteristics typical of a type II deiodinase. The levels of RC5'DII transcripts in R. catesbeiana tadpole tail and liver mRNA at stages XII and XXIII correspond well with that of type II deiodinase activity but not that of the type III activity in these tissues. These findings indicate that the amphibian type II 5'-deiodinase is a structurally unique member of the family of selenocysteine-containing deiodinases.

Amino Acid Sequence↗
PubMed1995

The type III 5-deiodinase in Rana catesbeiana tadpoles is encoded by a thyroid hormone-responsive gene.

We have recently reported that the Xenopus laevis complementary DNA (cDNA), XL-15, encodes a selenoprotein that is a 5-deiodinase (5D). XL-15 represents a gene that is up-regulated by thyroid hormone in this species. We now report the isolation from a Rana catesbeiana (RC) cDNA library of a cDNA for the RC 5D (RC5D). RC5D a 1534-base pair cDNA that exhibits 78% identity to XL-15 in the coding region, including the TGA codon, which in XL-15 encodes selenocysteine, and a putative selenocysteine insertion sequence (SECIS) located in the 3'-untranslated region. Transcripts of RC5D, synthesized in vitro, induce 5D activity after their injection into Xenopus laevis oocytes. RC5D hybridizes to a 2.2-kilobase messenger RNA (mRNA) species in RC tissues. The expression of RC5D was studied in 10 tissues from untreated and T3-treated premetamorphic tadpoles. RC5D mRNA transcripts and 5D activity were detected in most tissues examined, and the levels of both were greatly increased in T3-stimulated tadpoles. Furthermore, the levels of RC5D mRNA transcripts correlated closely with 5D, but not with 5'-deiodinase (5'D), activity. 5'D was not enhanced by a 4-day exposure of the tadpoles to T3. The up-regulation of RC5D gene expression by T3 was demonstrable in tadpoles by stage V, but was found to be transient. The levels of RC5D transcripts and 5D activity were highest after 2-3 days and 5 days of T3 exposure, respectively, but had fallen by 10 days to levels comparable to those in unexposed tadpoles. It is concluded that RC5D encodes a selenoprotein that is a 5D, and it represents a gene that is up-regulated by T3. On the basis of the findings presented herein, it is suggested that the 5D system plays a major role in regulating intracellular T3 levels in developing tadpoles.

Amino Acid Sequence↗
PubMed1995

Effect of glucocorticoids on thyroid hormone action in cultured red blood cells from Rana catesbeiana tadpoles.

Metamorphosis in anuran amphibia requires thyroid hormone (TH) and can be induced prematurely by the administration of TH. There is also evidence that the developmental effects of TH in these forms are modified by other hormones. For example, PRL has been shown to retard and corticosterone (B) to accelerate some, but not all, components of TH-induced metamorphosis. Red blood cells (RBCs) of Rana catesbeiana tadpoles exhibit a 4- to 5-fold increase in thyroid hormone receptor (TR) number (sites per nucleus) in vivo during either spontaneous or TH-induced metamorphosis. In the present study this TH-induced effect on RBC TR number was examined in an in vitro culture system. RBC TR number was increased by T3 in vitro; the maximum effect (2-fold increase) was obtained after exposure to 0.3 nM T3 for 60 h. This T3-induced increase in TR number was completely abolished in the presence of either 34 nM B or 10 nM dexamethasone, whereas basal TR number was unaffected. The effect appears to be a specific effect of glucocorticoid (GC), because it was not mimicked by the sex steroid, testosterone, and it was not obtained when RU-486, a glucocorticoid antagonist, was included with B in the medium. Other experiments demonstrated that the T3-induced increase in RBC TR was associated with an increase in the TR alpha messenger RNA level. This increase in TR alpha messenger RNA was reduced, but not eliminated, in the presence of concentrations of GC that abolished the TH-induced increase in TR, suggesting that the effects of GC occur in part at a pretranslational level. Using a GC binding assay, tadpole RBCs were found to contain approximately 10(4) GC receptors/cell. These findings indicate that B may be a physiological modulator of TH action in tadpole RBCs. This inhibitory effect of GC contrasts with previous reports that GC accelerates some of the morphological effects of TH in developing tadpoles, indicating that the nature of this modulating effect on TH action is tissue specific.

Animals↗
PubMed1994

A thyroid hormone-regulated gene in Xenopus laevis encodes a type III iodothyronine 5-deiodinase.

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in the mammalian enzyme. We report here that the protein encoded by the XL-15 cDNA efficiently catalyzes the (inner ring) 5-deiodination of 3,5,3'-triiodothyronine with a Km value of 2 nM and is resistant to inhibition by propylthiouracil and aurothioglucose. Our analysis confirms that the UGA codon encodes a selenocysteine that is critical for the catalytic activity of the enzyme. In addition, the direct induction of XL-15 mRNA levels by thyroid hormone in X. laevis tadpole tail tissue and cultured cell lines correlates closely with increases in 5- (but not 5'-) deiodinase activity. These findings indicate that the XL-15 cDNA encodes a type III 5-deiodinase and underscores the importance of the trace element selenium in thyroid hormone metabolism.

Amino Acid Sequence↗
PubMed1994

Cloning of a thyroid hormone-responsive Rana catesbeiana c-erbA-beta gene.

Two types of thyroid hormone receptor (c-erbA) gene have been identified in mammals and in lower species including chickens and the amphibian Xenopus laevis. The two genes are located on different chromosomes and have been named TR alpha and TR beta. We have described previously the cloning of a TR alpha cDNA from Rana catesbeiana (RC) tissues (RC15) and we now report the cloning of a TR beta cDNA from this species. The cloning strategy employed utilized the polymerase chain reaction (PCR), with primers based on the sequences of the X. laevis TR beta cDNA (XenTR beta) and an RCTR beta genomic clone, which, by analogy with XenTR beta, contains some of the 3' end of the open reading frame together with 3'-untranslated sequences. At the nucleotide and amino acid levels, respectively, the cloned RCTR beta cDNA is 90% and 98% homologous with XenTR beta, and 72% and 76% homologous with RC15. Following in vitro transcription and translation, the cDNA was shown to encode a 48 kilodalton protein which binds 3,5,3'-triiodothyronine (T3) with high affinity (mean Kd: 0.032 nM). Samples of total or poly(A) +RNA from tadpoles at different stages of metamorphosis and from adult frogs were analyzed for the presence of TR beta-specific transcripts by slot blot analysis using as probe a 258 bp section of the RCTR beta cDNA. This section of the cDNA does not hybridize to the corresponding section of RC15. In confirmation of previous findings, beta-specific transcripts were not detected in RNA from tadpole red blood cells (RBCs) and none was found in RBCs from adult frogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Rana catesbeiana tadpole red blood cells express an alpha, but not a beta, c-erbA gene.

Thyroid hormone (TH) receptor number in red blood cells (RBCs) from Rana catesbeiana (RC) tadpoles increases 4-fold during both spontaneous and TH-induced metamorphosis, an effect that we have previously shown to be preceded by an increase in the level of c-erbA-related mRNA. The goals of the present study were to obtain an RC c-erbA alpha cDNA that contains the entire open reading frame for a putative TH receptor protein, to determine if this protein has characteristics typical of a TH receptor, and to assess its contribution to the developmentally related increase in TH receptor number. To accomplish this, the missing 5'-sequence of a previously isolated partial RC c-erbA alpha cDNA (RC12) was synthesized by polymerase chain reaction (PCR) and spliced to RC12 to yield a 1490-basepair cDNA (RC15) that contained the entire coding sequence of the receptor protein. Transcription of RC15 followed by translation of its mRNA in a rabbit reticulolysate system yielded a 50-kilodalton protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds T3 with high affinity (Kd, approximately 0.1 nM), and its affinity for T3 is at least 5 times that for T4. The results of cotransfection studies indicate that RC15 can function as a TH receptor; when COS cells were cotransfected with a construct consisting of RC15 cloned in the expression vector CMV4 and TK28 mult, a construct containing rat GH gene TH response element sequences up-stream of a chloramphenicol acetyltransferase reporter gene, chloramphenicol acetyltransferase activity is expressed in the presence, but not in the absence, of T3. To determine whether RBCs contain any c-erbA beta mRNA transcripts that might contribute to the developmentally related increase in the transcripts detected using RC c-erbA alpha cDNAs, alpha- and beta-specific cDNAs were synthesized by PCR and used as probes in a variety of hybridization assays. In all experiments using conditions in which c-erbA beta transcripts were detectable in other tissues, there was no evidence that tadpole RBCs contained such species. Lack of any beta-specific transcripts was confirmed by PCR, using as template cDNA prepared by reverse transcription of RC RBC RNA. It was also noted that the RBC at metamorphic climax is the tissue with the highest content of alpha-specific c-erbA transcripts. It is concluded that the c-erbA alpha gene encodes a TH receptor, and that only the alpha-gene is expressed in tadpole RBCs and subject to regulation during development and by TH.

Amino Acid Sequence↗
PubMed1992

Thyroid hormone receptors and iodothyronine deiodinases in the developing Mexican axolotl, Ambystoma mexicanum.

The Mexican axolotl, Ambystoma mexicanum, is a neotenous salamander that rarely undergoes anatomical metamorphosis, but can be induced to do so by administration of thyroxine (T4). The neoteny appears to be due primarily to low levels of plasma T4 secondary to a low rate of secretion of thyroid-stimulating hormone. However, other factors may also be involved. In anuran amphibia, metamorphosis is accompanied by alterations in thyroid hormone receptor concentration and marked changes in the activities of the iodothyronine deiodinase systems, all of which contribute to enhancing peripheral sensitivity to circulating T4. The present study was designed to assess these functions in the tissues of the axolotl. Putative 3,5,3'-triiodothyronine (T3) receptors were readily detected in axolotl red blood cells, and the receptor number (sites/nucleus) showed a developmental decline, comparable to that seen in anuran amphibia, as the larval cells with a high receptor number were replaced with adult cells with a low receptor number. Saturable T3 nuclear binding sites were also detected in liver, but the receptor number was too low to quantitate. Using a sensitive RIA, T4 and T3 were below detectable levels in juvenile axolotls and T4 was just detectable in some of the adults. Following injection of [125I]T4, [125I]T3 was detected in plasma and liver of adult, but not of juvenile, axolotls. Some 5'-deiodinase activity was consistently detected in preparations of larval skin, and in adult skin and gut, but it was too low to permit kinetic analyses. Activity was not increased following treatment with sufficient T4 to induce anatomical metamorphosis. 5-Deiodinase activity was not detected in any tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗