Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Characterization of Rana catesbeiana HSP30 gene and its expression in the liver of this amphibian during both spontaneous and thyroid hormone-induced metamorphosis

During metamorphosis, the Rana catesbeiana tadpole undergoes developmental changes in almost every tissue/organ. These changes prepare the ammonotelic, swimming larva for its transition to a ureotelic, terrestrial adult, and involve dramatic remodeling. The postembryonic changes in this tadpole are initiated by the thyroid hormones (TH) and result in the extensive degradation of proteins and degeneration of tissues characteristic of the larval phenotype and in the de novo synthesis of proteins characteristic of the adult phenotype. We questioned whether the drastic nature and abruptness of the TH-dependent, postembryonic changes occurring in the tissues of this tadpole might be perceived by the cells in some tissues as stressful and, therefore, cause them to express heat shock and/or stress-like proteins. To address this question, we isolated and characterized a Rana catesbeiana hsp30 gene and used sequences from it to determine if mRNAs encoded from it, or other members of this gene family, are expressed in tissues of tadpoles undergoing metamorphosis. Our results demonstrate that the liver of metamorphosing Rana catesbeiana tadpoles accumulate hsp30 mRNAs and express the heat shock proteins they encode. The fact that the expression of these hsp30s in the liver of these tadpoles is coincidental with the TH-induced expression of genes encoding the liver-specific urea cycle enzymes suggests that TH may influence, directly or indirectly, the expression of these hsp30 genes and, moreover, implies that the presence of one or more of these heat shock proteins may be necessary for the developmental transitions occurring in this organ. © 1996 Wiley-Liss, Inc.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 2. Response to season,temperature and thyroid hormone in Rana pipiens

The impact of season and temperature on frog liver γ-glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short-term (3 weeks) temperature acclimation; and subjected to multiple-temperature shifts. Plasma levels of T₃ were determined. γ-Glutamyltranspeptidase was found to be 2·2-fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1·7-fold in the winter frog by warm acclimation; and increased by 1·9-fold in the summer frog and 2·8-fold in the winter frog subjected to multiple-temperature shifts. Plasma T₃ levels were found to be 42-fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2·9-fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T₃ replacement during the last phase of the multiple-temperature shift protocol, restored the plasma T₃ levels to 75 percent of the control levels and prevented the increase evoked by the multiple-temperature shifts in γ-glutamyl-transpeptidase activity. Indeed, enzyme activity in the T₃ replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.