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F E Carr

Publications and source records attributed to F E Carr.

At least 37 records · Page 2Linked to original sources

Lymphocytes express thyrotropin receptor-specific mRNA as detected by the PCR technique.

Human lymphocytes are known to play a critical role in autoimmune diseases both by producing antibodies and by participating in lymphokine-cellular interactions. TSH, a classic pituitary hormone, may be secreted by human lymphocytes, and controversy has existed whether a specific, authentic TSH receptor also was present on the surface of these cells. The objective of our study was to identify TSH receptor transcripts after designing specific oligonucleotides that would recognize a unique putative TSH binding area of the thyroidal TSH receptor. The existence of TSH receptor transcripts was probed by employing these primers in a PCR reaction with cDNA derived from normal peripheral human lymphocytes and human thyroid tissue, as well as with cDNA from a medullary cancer cell line and rat liver. Human lymphocytes and thyroid tissue, but not medullary cancer cells or rat liver, demonstrated specific TSH receptor amplification product both by ethidium bromide staining and by Southern blot hybridization with labeled TSH receptor cDNA. The lymphocyte cDNA was partially sequenced and found to be identical to the thyroid-derived cDNA. These findings indicate that normal, nonactivated, human lymphocytes produce transcript for a TSH receptor that appears identical to that in thyroid tissue. Future studies should focus on the regulation of this transcript, as well as on the role TSH and TSH receptor may play in modulating local lymphokine activation of T and B cells, both in normal conditions and in autoimmune thyroid disease.

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Effects of irradiation and semistarvation on rat thyrotropin beta subunit messenger ribonucleic acid, pituitary thyrotropin content, and thyroid hormone levels.

The effect of radiation-induced anorexia on serum thyrotropin (TSH), pituitary TSH-beta mRNA, pituitary TSH content, serum thyroxine (T4), and serum 3,5,3'-triiodothyronine (T3) was investigated using feed-matched controls. Rats received 10 Gy gamma whole-body irradiation and were examined 1-3 days postirradiation. Feed-matched and untreated controls were also studied. The average food intake of the irradiated and feed-matched groups was approximately 18% of the untreated controls. Over the three day period both the irradiated and feed-matched groups lost a significant amount of body weight. The serum T4 levels of both the irradiated and feed-matched groups were not significantly different from each other, but were significantly depressed when compared to the untreated control group. The serum TSH and T3 were, however, significantly greater in the irradiated than the feed-matched groups at day 3 posttreatment. To determine if the difference in the serum TSH level between the two groups was due to a pretranslational alteration in TSH production, we measured the TSH-beta mRNA using an RNA blot hybridization assay. We found that the TSH-beta mRNA level was the same in the irradiated and feed-matched groups, suggesting that the mechanism responsible for the radiation-induced increase in the serum TSH level is posttranscriptional. Pituitary TSH content in the irradiated rats was significantly less than in pair-fed controls, suggesting that irradiation may permit enhanced secretion of stored hormone.

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Thyroid hormone regulation of the rat glycoprotein hormone alpha-subunit gene promoter activity.

An important physiological control of the glycoprotein hormone alpha-subunit is the negative feedback by thyroid hormones in the thyrotrope. A region of the rat glycoprotein hormone alpha-subunit gene that is involved in transcriptional regulation by thyroid hormone has been identified by transient transfection studies, and sequence-specific binding of the thyroid hormone receptor to a site within this region has been demonstrated. Deletion-mutation studies using plasmid expression vectors containing either 246, 170, or 80 base pairs of the 5'-flanking region of the rat alpha-subunit gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene demonstrate 3,5,3'-triiodo-L-thyronine (T3)-regulated expression in GH3 cells, a T3-responsive somatotrophic cell line. In order to investigate the possibility of thyroid hormone receptor interaction with this segment of the rat alpha-subunit gene, the binding of the thyroid hormone receptor to synthetic oligodeoxynucleotides was analyzed using an avidin-biotin complex DNA binding assay. An oligodeoxyribonucleotide representing a fragment of the alpha-subunit gene from -74 to -38, relative to the transcriptional start site, shows significant binding to [125I]T3-receptor complex present in nuclear extracts of GH3 cells. This fragment binds receptor to a degree similar to that seen with a fragment of the rat growth hormone gene which contains a putative thyroid hormone-responsive element. In addition, this fragment of the rat alpha-subunit gene binds to the in vitro synthesized human c-erbA beta protein, which has been identified as a member of the family of putative T3 receptors. These data demonstrate that a cis-active thyroid hormone-responsive element resides in the 5'-flanking region of the rat alpha-subunit gene and that the mechanism involved in the suppression of expression of this gene by T3 could involve specific binding of the thyroid hormone receptor to this region of the gene.

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Thyroid hormones regulate rat thyrotropin beta gene promoter activity expressed in GH3 cells.

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSH beta DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH3, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T3 (10(-11) M to 10(-7) M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10(-8) M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T3 was equivalent even in the 57 bp construct. In contrast, T3 had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSH beta gene 5'-flanking region can direct heterologous gene expression in GH3 cells and contains sequences which have properties of a putative cis-active T3 responsive regulatory element(s).2+he

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Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells.

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

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Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization.

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.

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Isolation and characterization of the rat thyrotropin beta-subunit gene. Differential regulation of two transcriptional start sites by thyroid hormone.

The gene encoding the beta-subunit of rat thyrotropin (TSH beta) has been isolated and characterized. Blot hybridization of restriction enzyme digests of rat genomic DNA suggests that the gene is present in a single copy. The transcriptional unit is 4.9 kilobases in size representing 3 exons interrupted by 2 introns of 3.9 and 0.4 kilobases. Its nucleotide sequence reveals that the locations of the exon/intron junctions are one nucleotide upstream from the translational start and between codons +34 and +35. The location of the second intron is apparently strictly conserved among the glycoprotein hormone beta-subunit genes being four codons downstream from a region encoding a consensus sequence: Cys-Ala-Gly-Tyr. Using S1 nuclease mapping and oligonucleotide-primed reverse transcription of normal and thyroidectomized rat pituitary mRNA, two transcriptional start sites were identified in the rat TSH beta gene that are 28 and 71 nucleotides upstream from the translational start site. The level of TSH beta mRNA containing the downstream site is altered by thyroidal status whereas the other mRNA utilizing the upstream cap site appears to be constitutively expressed. Characteristic promoter elements are present in the 5'-flanking region including TATAAA or Goldberg-Hogness consensus regions which are present 29 and 26 bases upstream from the respective starts of transcription. Also, several CAAT boxlike sequences are located between 95 and 300 bases upstream from the start of translation. Isolation and characterization of the gene encoding the TSH beta gene will facilitate the study of the molecular mechanisms by which hormones regulate TSH beta gene expression.

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Thyroid hormone regulation of the rat thyrotropin beta-subunit gene.

In order to determine the molecular mechanisms by which thyroid hormone and other factors regulate the biosynthesis of thyrotropin (TSH), we have isolated and characterized the gene encoding the beta-subunit of rat TSH. The transcriptional unit is 4.9 Kb in size and contains 3 exons and two introns of 3.9 and 0.4 Kb. The gene possesses two promoters and hence two associated transcriptional start sites. Several lines of evidence indicate that there is differential regulation of the two TSH beta transcripts by thyroid hormone. In particular, rat TSH beta mRNA-2, the transcript associated with the downstream promoter, is regulated 6- to 8-fold by thyroid hormone. Further studies involving in vitro mutagenesis and transfection of the TSH beta gene into pituitary cells will hopefully provide important information concerning structure-function relations of the regulatory regions of the gene and thyroid hormone action.

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Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation.

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.

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Hepatic messenger ribonucleic acid activity profile of rats subjected to alterations in thyroidal and adrenocortical states: evidence for significant interaction.

We have previously established the value of 2-dimensional electrophoretic mRNA activity profiles for investigating the hepatic genomic response to several metabolic perturbations, such as thyroid hormone or GH treatment, diabetes, high carbohydrate diet, starvation, and uremia. We now report the effects of adrenalectomy and dexamethasone treatment, and compare these with alterations due to thyroidectomy and T3 treatment. Total rat hepatic RNA was isolated and translated in a reticulocyte lysate system. The [35S]methionine-labeled translated products were separated by 2-dimensional gel electrophoresis and quantified with computerized videodensitometry. Of 200 consistently quantifiable products, 14 (7%) were altered by adrenalectomy and dexamethasone, including 4 products (46, 47, 56, and 57) which have not been observed to change in previous studies from this laboratory. Adrenalectomy increased 5 and decreased 2 products, whereas dexamethasone increased 1 and decreased 8 products. Two products maintained the same directional shift in the transitions form adrenalectomy to control and from control to the dexamethasone-treated state. Thyroidectomy and T3 altered 13 products. Thyroidectomy increased 2 and decreased 7 products, whereas T3 treatment increased 6 and decreased 3 products. Four products maintained the same directional shift in the transitions from thyroidectomy to control and from control to the T3-treated state. In all of the manipulations performed (adrenalectomy, thyroidectomy, dexamethasone treatment, and T3 treatment), a total of 20 separate products changed. One third were affected by alterations of both the steroidal and thyroidal states. However, when adrenalectomy and thyroidectomy were compared, only 7% of the shifts were concordant, whereas 30% of the shifts were concordant when treatment with dexamethasone and T3 were compared. These results demonstrate that the mRNA activity response is highly specific for each hormonal manipulation. In addition, unanticipated interrelationships between steroidal and thyroidal states were observed. In some, the presence of T3 appears necessary for the suppressive effect of dexamethasone. Others show that T3 appears to inhibit a stimulatory effect of dexamethasone. Specificity of response to dexamethasone is emphasized by the lack of response to vitamin D, deoxycorticosterone, and dihydrotestosterone and by a different response to estradiol from dexamethasone.

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Rapid simultaneous measurement of rat alpha- and thyrotropin (TSH) beta-subunit messenger ribonucleic acids (mRNAs) by solution hybridization: regulation of TSH subunit mRNAs by thyroid hormones.

Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.

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Quantitative investigation of hepatic genomic response to hormonal and pathophysiological stimuli by multivariate analysis of two-dimensional mRNA activity profiles.

We have applied techniques of multivariate analysis to the characterization and comparison of the effects of various pathophysiological and hormonal stimuli on the expression of the rat hepatic genome at a pretranslational level. In vitro translated products were resolved by two-dimensional gel electrophoresis. We analyzed 10 pathophysiological states brought about by variation in thyroidal status, starvation, administration of high carbohydrate diet, and the production of experimentally induced diabetes mellitus. Each state differed significantly from every other state in the two-dimensional electrophoretic profiles. The set consisting of the minimal number of products necessary for maintaining the distinctive patterns was identified. The analysis also defined those clusters of products that behaved in a coordinate fashion in response to the various stimuli. Lastly, the similarity and dissimilarity of hepatic mRNA activity profiles to each other could be geometrically represented in three-dimensional space. Our finding that the hepatic mRNA activity profile could distinguish reliably between closely related hormonal and pathophysiological stimuli indicates the specificity of hepatic genomic expression. A systematic analysis of such profiles may be useful as an overall index of the biologic response at the hepatocellular level.

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Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.

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Distribution of thyroid hormone-responsive translated products in rat liver polysome and postribosomal ribonucleoprotein populations.

We investigated the possibility that hypothyroidism, like starvation, leads to a major disaggregation of polyribosomes and redistribution of mRNA to the nonpolysomal mRNA particle (mRNP) pool. The analysis involved the use of a translational assay of hepatic RNA obtained from bound and free polysomes and the subribosomal fractions (mRNP) of euthyroid and hypothyroid rats. Two-dimensional gel electrophoretograms of the in vitro synthesized 35S-labeled proteins revealed distinct protein patterns associated with each subcellular compartment. Of the 18 quantified T3-responsive sequences, the activities of 7 were preferentially associated with bound polysomes, 9 with free polysomes, and 2 with subribosomal mRNP in euthyroid rat livers. No major redistribution occurred in the transition from the euthyroid to the hypothyroid state. The distribution of mRNA activity of 1 T3-responsive sequence, that coding for Spot 14 (mRNAS14), was compared with the distribution of hybridizable mRNA, as determined with the use of a specific cDNA probe. In the euthyroid state, both techniques showed that approximately 70% of the total hepatic mRNAS14 was associated with free polysomes and 30% was associated with subribosomal complexes. A similar distribution was characteristic of the hypothyroid state. These findings demonstrate that T3-responsive sequences are found in all three mRNA compartments and that thyroid hormone action is not typically associated with a redistribution of mRNA among subcellular pools.

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Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.

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Effects of triiodothyronine and glucose on cultured rat hepatocyte gene expression.

Both thyroid hormone and carbohydrate feeding are known to influence hepatic gene expression in vivo. In order to determine whether these compounds act directly on the liver, we have utilized an isolated adult rat hepatocyte culture to examine the in vitro influence of either T3 or glucose on a wide range of mRNA activities. RNA was extracted from hepatocyte cultures, translated in the rabbit reticulocyte system, and the translated products separated by two-dimensional gel electrophoresis. We have found a strong similarity between the hepatic mRNA response to T3 administered either to the animal or added to the culture medium and between the response to high carbohydrate feeding and an increase in the medium glucose concentration. Our studies suggest that the isolated hepatocyte culture is a useful model for the study of the influence of hormones and metabolites on the expression of specific mRNA's.

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Characterization of solubilized prolactin receptors from Rana catesbeiana tadpole tissues.

Prolactin (PRL) receptors were solubilized from Rana catesbeiana tadpole liver and tail fin and female Sprague-Dawley rat-liver membranes by treatment with 1% Triton X-100. Binding of [125I]oPRL to tadpole-liver and tail-fin solubilized extracts reached equilibrium by 12 h at 19 degrees C. The binding was linear up to 50 micrograms of tadpole liver and tail-fin protein and 30-40 micrograms of rat-liver protein. Solubilization did not affect the dissociation constant for [125I]oPRL binding but did result in a greater number of sites. The binding was specific for oPRL and hGH, which has PRL-like activity. Neuraminidase pretreatment of membranes altered the binding affinity of oPRL to tadpole-liver membranes and solubilized extracts but not to tadpole-tail fin membranes or extracts. Pretreatment of membranes with neuraminidase did not affect the binding capacity of tadpole-liver or tail-fin membranes or solubilized extracts.

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Changes in specific prolactin binding in Rana catesbeiana Tadpole tissues during metamorphosis and following prolactin and thyroid-hormone treatment.

The prolactin-binding affinity (KD) and number of binding sites *N) in Rana catesbeiana tadpole liver, tail fin and kidney tissues were studied during metamorphosis and following administration of oPRL and L-T3 to premetamorphic tadpoles. With increasing developmental stage there was an increase in N; a maximum was found at stage XVIII followed by a gradual decrease in N through metamorphic climax for all 3 tissues. No change in KD was noted. L-T3 treatment of premetamorphic tadpoles for 7 days caused a significant decrease in tail length and height and body length and an increase in hindlimb length with a concurrent increase in N of approximately 3-fold while treatment for 1 or 3 days was without effect on tadpole morphology or oPRL binding. OPRL treatment for 7 days caused a significant increase in tail length and height and body length with no significant changes in hindlimb length and a 3-5-fold increase in N. Treatment with both L-T3 and oPRL for 7 days resulted in an inhibition of the T3-induced decrease in tail length and height and body length and no inhibition of the hindlimb length increase. N increased in all tissues similar to that found with eight treatment alone. No change in KD was noted in any of these studies. Therefore, oPRL and L-T3 are able to regulate the numbers of specific oPRL-binding sites in amphibian tissues. The change in N with development parallels the reported change in tadpole pituitary capacity to stimulate growth but occurs prior to the reported surge of endogenous T3 during metamorphosis. Thus, the variation in the number of oPRL-binding sites may be due to the changes in endogenous PRL levels during development.

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