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J Ruel

Publications and source records attributed to J Ruel.

10 recordsLinked to original sources
PubMed1988

A monoclonal antibody to the rat nuclear triiodothyronine receptor: production and characterization.

The nuclear T3 receptor (NTR) was affinity-labeled with bromoacetyl-[125I]T3, purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and used to immunize BALB/c mice. Spleen cells from one strongly immunoreactive mouse were fused with Sp2 mouse myeloma cells, and 328 hybridomas were screened by a dot-blot immunoassay using as antigen, a preparation of NTR partially purified by diethylaminoethyl-Sephadex chromatography. Four positive cultures were thus found; three of which were confirmed by comparing Western blotting patterns with the electrophoretic mobility of the affinity-labeled NTR. One of these 3 hybridomas was further subcloned by limiting dilution and gave rise to the 2B3 clone, which produces an immunoglobulin of the immunoglobulin G1 subclass. Several lines of evidence indicated that the 2B3 monoclonal antibody was indeed directed against the NTR. The antibody recognized a protein with the same electrophoretic mobility as the affinity-labeled receptor. Thus, Western blotting revealed a predominant protein with a mol wt of 57,000 and a less abundant 45,000 component on sodium dodecyl sulfate gels, and multiple isoelectric variants of the 57,000 protein, with a predominant form at pI 6.2, were detected on two-dimensional gels. Incubation of the 2B3 antibody with the NTR labeled with [125I]T3 resulted in the formation of an antibody-receptor complex, as indicated by a shift of the radioactivity peak upon gel filtration on Sephacryl S-300. In contrast, control ascitic fluid did not change the elution profile of the labeled NTR. The 2B3 antibody is able to remove the T3-binding activity from rat liver nuclear extracts. Finally, in accordance with previous T3-binding experiments, expected amounts of NTR were found in pituitary, liver, brain, kidney, spleen, and testis with the use of the Western blotting technique and immunohistochemistry on frozen tissue sections. This antibody should prove useful in the characterization and purification of the NTR and also in the study of its distribution in different tissues and cell types.

Animals↗
PubMed1987

Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules.

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.

Amino Acid Sequence↗
PubMed1987

Thyroid hormones and brain development.

We have limited ourselves to the deleterious effects of the absence of thyroid hormones on the development of the central nervous system and have not discussed the problems caused by hyperthyroidism. A short "critical period" during which thyroid hormones are essential for normal maturation of the CNS is evident at anatomical, biochemical, and neurophysiological levels. In the last decade we have made progress toward understanding the mechanism of action of thyroid hormones, due in part to numerous studies of the ontogenesis and distribution of the nuclear T3 receptor. These studies can indicate where the molecular events that control the growth and maturation of the brain are initiated. However, much further research in this area is needed to comprehend further the relation between thyroid hormones and brain development.

Aging↗
PubMed1986

Regulation of protein phosphorylation by triiodothyronine (T3) in neural cell cultures. Part I: Astrocytes.

Dissociated cells from 2-day-old rat cerebral hemispheres were cultured for 17 days in absence of thyroid hormones using conditions yielding mainly glial cells. Triiodothyronine (10(-8) M) was added for 0-72 h before the end of the incubation and [32P]phosphate was added for the last 4 h. Soluble (105,000 X g supernatant), particulate (105,000 X g pellet) and HMG (high mobility group; 0.75 M perchloric acid-soluble proteins) fractions were prepared and phosphorylated proteins in each fraction were analyzed by polyacrylamide gel electrophoresis. In the soluble fraction a protein (Mr = 19,000) incorporates less [32P]phosphate after only 4 h of T3 treatment. The maximal effect is attained after 7 h (-42%) and remains unchanged up until 72 h. In this fraction, the phosphorylation of some other proteins is increased but the maximal effect is observed 48 and 72 h after T3 administration. In the particulate fraction, exposure to T3 rapidly (4 h) increases the amount of a protein (Mr = 45,000) identified as beta-actin. Protein phosphorylation in this fraction is slightly, or not at all, affected by T3. In contrast, a rapid (between 4 and 7 h) increased phosphorylation of a 17 kDa protein in the HMG fraction is observed following T3 stimulation. This nuclear protein was further characterized as HMG 14. These results show that thyroid hormones can produce direct effects (not mediated by neurons) on the phosphorylation of specific proteins in cultured glial cells. Possible functional implications of the observed protein changes are discussed in this paper.

Actins↗
PubMed1986

Regulation of protein phosphorylation by triiodothyronine (T3) in neural cell cultures. Part II: Neurons.

Cerebral hemisphere from 16- to 18-day-old rat fetuses were dissociated and cells were cultured in absence of thyroid hormones. Neuron-enriched cultures were obtained either by using cells after 6 days of culture (before extensive glial cell proliferation) or by adding cytosine arabinoside for 48 h after 4 days of culture and using cells on day 9. Cells were incubated with T3 (10(-8) M) for 0-72 h and [32P]phosphate was added for the last 4 h of incubation. HMG (high mobility group; 0.75 M perchloric acid-soluble proteins) were prepared and phosphorylated proteins were analyzed by polyacrylamide gel electrophoresis. T3 rapidly (4-7 h) increased the phosphorylation of histone H1 and of a protein with apparent molecular mass of 17000 Da identified as HMG 14. In addition, in cells not treated with cytosine arabinoside, histone H1 was resolved into 3 subfractions and each of these responded to the hormone with a different time course. These results indicate that thyroid hormones act on the phosphorylation of specific nuclear proteins and therefore may influence chromatin structure and gene expression in primary neuronal cell cultures.

Animals↗
PubMed1986

Production of an antibody against rat liver nuclear T3 receptor.

Rabbits were immunized with rat liver nuclear L-triiodothyronine (T3) receptor purified by preparative sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis using bromoacetyl[125I]T3 as an affinity label. SDS-PAGE confirmed the presence of two receptor forms of the apparent molecular weights 57,000 and 45,000. We describe here a specific antibody, raised against the 57,000 receptor type, which reacts with both receptor forms as assessed by electroimmunoblotting and immunoprecipitation in liquid medium.

Animals↗
PubMed1985

Regional distribution of nuclear T3 receptors in rat brain and evidence for preferential localization in neurons.

We examined the distribution of nuclear T3 in mature rat brain with the aim of determining specific targets of thyroid hormones within this tissue. Saturation experiments, performed in 9 different structures of the brain and in 4 parts of the cortex, revealed the presence of a single class of binding sites with a mean Ka of 0.53 X 10(10) M-1. The highest concentrations of receptors were found in the amygdala (0.523 +/- 0.025 ng T3/mg DNA, Mean +/- SE) and the hippocampus (0.438 +/- 0.071 ng T3/mg DNA) while the lowest were in the brain stem (0.058 +/- 0.003 ng T3/mg DNA) and the cerebellum (0.079 +/- 0.026 ng T3/ml DNA). The receptor was not uniformally distributed within the cerebral cortex, its concentration being relatively high in the central sections and intermediate in the remaining portions. The cell type distribution of the T3 receptor was studied by separating glial and neuronal nuclei on a discontinuous sucrose gradient. There was no detectable specific T3 binding in the fraction of oligodendrocyte nuclei (approximately 95% pure). Conversely, the neuron-enriched fraction (approximately 60%) showed a significant increase in receptor concentration compared to total nuclei (35-40% neurons): 0.857 +/- 0.196 vs 0.511 +/- 0.095 ng T3/mg DNA (p less than 0.01) in the cortex and 0.425 +/- 0.018 vs 0.234 +/- 0.24 ng T3/mg DNA (p less than 0.01) in the forebrain. The absence of nuclear T3 receptors in oligodendrocytes may have important implications on the mechanism of action of thyroid hormone in myelination.

Animals↗
PubMed1985

Triiodothyronine increases glutamine synthetase activity in primary cultures of rat cerebellum.

Dissociated cells from 2-3 day-old rat cerebella were cultured in absence of thyroid hormones using conditions yielding mainly glial cells. After 7, 14 and 21 days in vitro, triiodothyronine (60 nM) was added to a set of dishes and glutamine synthetase activity was measured after 24, 48, and 72 h in both control and triiodothyronine-treated cultures. Basal glutamine synthetase activity increased more than 6 X between 7 and 21 days of culture. Triiodothyronine produced significant increases of glutamine synthetase activity after 72 h in 7-day-old cultures (+ 16%), after 48 h in 14-day-old cultures (+ 45%) and after 24 h in 21-day-old cultures (+ 27%). This effect depends on the initial plating density and is not observed if cells are plated at less than 1 cerebellum equivalent per 60 mm dish. Dose-response experiments indicated that 10(-8) M of triiodothyronine induces maximal response whereas half-maximal response is achieved around 10(-10) M. These results show that physiological amounts of thyroid hormone can influence the maturation of astrocytes in culture.

Animals↗