Characterization of an iodothyronine 5'-deiodinase in gilthead seabream (Sparus auratus) that is inhibited by dithiothreitol.

Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T(4) to T(3) by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T(3) (rT(3)) ORD by 70 and 100% in kidney homogenates (IC(50) 0.4 mmol/liter) and microsomes (IC(50) 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 mumol/liter rT(3) and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology (> or = 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT(3) ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.

Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA.

In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken pre-proTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide. Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5' untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

Corticosterone-induced negative feedback mechanisms within the hypothalamo-pituitary-adrenal axis of the chicken.

This paper reports the results of in vivo and in vitro experiments on the feedback effects of corticosterone on the hypothalamo-pituitary-adrenal axis in embryos at day 18 of incubation and in 9-day-old chickens. In vivo, a significant negative feedback was detected on the levels of corticotropin-releasing factor (CRF) precursor (proCRF) mRNA and on the plasma concentration of corticosterone, two hours after a single intravenous injection with 40 microg corticosterone. In contrast, the levels of CRF peptide in the hypothalamic area, the CRF receptor type 1 (CRF-R1) mRNA and pro-opiomelanocortin (POMC) mRNA levels in the pituitary were not affected by the in vivo administration of corticosterone. In vitro, incubation with 1 microM corticosterone did not affect the CRF-R1 mRNA levels in the pituitary, but significant feedback inhibition was observed on the POMC mRNA levels. These in vitro effects were the same at the two ages studied. The in vitro feedback effect on the proCRF gene expression, however, differed with age. In 9-day-old animals a decrease in gene expression was observed which was not detectable in embryonic tissue at day 18 of the ontogeny.