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J H Dussault

Publications and source records attributed to J H Dussault.

At least 37 records · Page 2Linked to original sources
PubMed1992

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.

Animals↗
PubMed1991

Immunocytochemical localization of thyroid hormone receptors in the adult rat brain.

It is generally accepted that thyroid hormones act at the genomic level through an interaction with specific nuclear receptors. Using a monoclonal antibody raised against the rat liver nuclear L-T3 receptor (NTR), we report here the immunocytochemical localization of T3 receptors in the adult rat brain. The strongest NTR immunoreactivity was found in the olfactory bulb, the hippocampus, the dentate gyrus, the amygdala areas, and the neocortex (layers III-VI). An intermediate NTR immunoreactivity was found in the hypothalamus, whereas the thalamus, the caudate-putamen, and the pallidum were weakly NTR-immunoreactive. In the cerebellum, a strong NTR immunoreactivity was found in the nuclei of Purkinje cells, in the internal granular layer, and in some nuclei of cells located in the molecular layer. In the brainstem, a strong NTR immunoreactivity was found in the lateral mamillary nucleus and the interstitial nucleus. A weak to moderate NTR immunoreactivity was observed in the central gray matter, while the substantia nigra and the interpeduncular nucleus were weakly stained. Furthermore, we also found NTR immunoreactivity in the nuclei of ependymocytes, epithelial cells of the choroid plexus, and cells located in the white matter. At the electron microscope level, we confirm that the immunoreactivity was not only localized in the nuclei of neurons but also in the nuclei of astrocytes and medium oligodendrocytes. This study provides new information concerning the distribution of NTR in the rat brain: (1) NTR are present not only in neurons but also in glial and ependymal cells, and (2) there is a regional and cellular heterogeneity in the distribution of NTR in the central nervous system.

Animals↗
PubMed1991

Production of a specific polyclonal antibody against the rat beta thyroid receptor, using synthetic peptide as antigen.

We have developed a polyclonal antibody to a synthetic peptide corresponding to the deduced amino acid residues 67-80 of the amino terminal sequence of the rat thyroid receptor beta cDNA. Immunopositive sera were identified by radioimmunoassay and purified by affinity chromatography. The antibody specifically immunoprecipitated the labelled thyroid receptor beta synthetized in vitro but not the rat thyroid receptor alpha. Furthermore, 38% of the L-T3 binding capacity of the rat liver nuclear receptor were immunoabsorbed by this antibody. By immunocytochemistry, this antibody stained specifically the nuclei of hepatocytes while no staining was observed in the rat testis. This study presents a specific polyclonal antibody against the thyroid receptor beta. This antibody will be of a great help in determining the role of the beta receptors in the mechanism of action of thyroid hormone.

Animals↗
PubMed1990

Influence of soluble environmental factors on the development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium: comparison with the effects of L-triiodothyronine (L-T3).

In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of L-triiodothyronine (L-T3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of L-T3 (CM + L-T3 or CM-L-T3). laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with L-T3. Furthermore, L-T3, NGF, CMs, laminin and FGF did not affect AChE+ cell survival, but significantly increased neurite outgrowth and branching with NGF and L-T3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of L-T3 with either laminin or FGF in culture, caused an additive effect of L-T2 in the neurite density of AChE+ cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated. and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1990

Immunocytochemical localization of thyroid hormone nuclear receptors in cultured acetylcholinesterase-positive neurons: a correlation between the presence of thyroid hormone nuclear receptors and L-tri-iodothyronine morphological effects.

A monoclonal antibody against the rat liver L-tri-iodothyronine nuclear receptor and acetylcholinesterase cytochemistry were used for the localization of thyroid hormone nuclear receptors in acetylcholinesterase-positive cell nuclei in fetal rat cerebral hemisphere neuronal cultures. After 3 days in vitro, the ratio of acetylcholinesterase-positive cells that were immunoreactive for the thyroid hormone nuclear receptor to those not stained for this receptor (74-26%, respectively) remains unchanged despite an increase in the number of acetylcholinesterase-positive cells with time (from day 3 to day 21) in culture. Furthermore, the addition of 3 X 10(-8) L-tri-iodothyronine in culture did not modify this ratio or have an effect on the number of acetylcholinesterase-positive cells, but significantly increased the neurite density in those acetylcholinesterase-positive cells that were immunoreactive for the thyroid hormone receptor. Conversely, no difference in the neurite densities of those acetylcholinesterase-positive cells not stained for this receptor was observed when cultured in the presence or absence of thyroid hormone. In other experiments with the same fetal brain cultures, treatment of cultures for 8 days with L-tri-iodothyronine, beginning on culture day 20, demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of L-tri-iodothyronine on immunoreactive acetylcholinesterase-positive cell neurite density is lost after 20 days in vitro. These results demonstrate, for the first time, the presence of L-tri-iodothyronine nuclear receptors in fetal rat acetylcholinesterase-positive neurons and the existence of a cellular heterogeneity in the distribution of the thyroid hormone receptor. The presence of these receptors in fetal brain acetylcholinesterase-positive neurons suggests that some effects of L-tri-iodothyronine on the maturation of a subpopulation of acetylcholinesterase-positive neurons may result from a direct effect of this hormone through an interaction with its specific nuclear receptors.

Acetylcholinesterase↗
PubMed1990

Interaction of sodium molybdate with the thyroid hormone receptor.

The 3,5,3'-triiodothyronine (T3) binding activity of solubilized nuclear proteins from rat liver was decreased when molybdate (10 mM) was present in the incubation medium in the absence of thiol reagents. The equilibrium affinity constant was reduced by 40%. The rate of degradation of T3-receptor complexes at 37 degrees C remained unchanged, but when the extracts were further reincubated in the presence of beta-mercaptoethanol, molybdate had a protective effect after 5 h incubation at 37 degrees C. In contrast, the thyroxine (T4) binding activity was not affected by heating at 37 degrees C or by molybdate. Ion-exchange chromatography confirmed the existence of a molybdate-receptor interaction: the T3-receptor complexes shifted from elution at 0.22 to 0.20 M NaCl with the progressive appearance of a small leader peak, whereas the T4-receptor complexes eluted in a large and split peak (0.22-0.4 M NaCl). The destabilizing effect on T3 binding induced by exogenous dephosphorylation is more efficiently reversed by beta-mercaptoethanol when the extracts were pretreated by molybdate. In controls, the loss of saturable T3 binding activity was recovered by 50% at a 10 mM concentration of beta-mercaptoethanol, but in the presence of molybdate, the loss of T3 binding activity was recovered by 50% at a 5 mM concentration of beta-mercaptoethanol. This molybdate-receptor interaction is similar to that with nuclear receptor models in term of (i) stabilization of hormone binding, (ii) dependency on a thiol, and (iii) reversibility of the destabilizing effect by exogenous dephosphorylation.

Alkaline Phosphatase↗
PubMed1989

Localization of c-ERB A proteins in rat liver using monoclonal antibodies.

Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction. These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses. In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts. Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized.

Animals↗
PubMed1989

Immunocytochemical localization of nuclear 3,5,3'-triiodothyronine (L-T3) receptors in astrocyte cultures.

By means of a monoclonal antibody (mab) against the rat liver nuclear L-T3 receptor (NT3R) and a polyclonal anti-GFAp serum, it has been possible to demonstrate nuclear thyroid hormone receptors in astrocyte cultures. On day 3, 47% of GFAp+ cell nuclei were labeled by 2B3 mab. Between day 3 and day 15, the number of GFA+ cell nuclei stained by 2B3 mab increased from 47 to 75%. Thyroid hormone nuclear receptors were present in fibrous and protoplasmic astrocytes. However, they developed asynchronously in both types of astrocytes. Indeed, 60% of fibrous astrocytes were stained by 2B3 mab on day 3 and this percentage reached 77% after 8 days in vitro. In contrast, only 30% of protoplasmic astrocytes were immunoreactive for 2B3 mab on day 3 and this percentage increased slowly reaching 47% on day 8 and around 75-80% on day 15. By immunoblotting, the monoclonal antibody recognized two bands of proteins with a molecular weight of 57 and 45 kDa respectively. These proteins have the same electrophoretic mobility as [125I]bromoacetyl-LT3 rat liver nuclear L-T3 receptor. This paper presents the first immunocytochemical localization of nuclear L-T3 receptors in astrocyte cultures. Furthermore, we show that thyroid hormone receptors develop more rapidly in fibrous than in protoplasmic astrocytes.

Animals↗
PubMed1989

Immunocytochemical localization of thyroid hormone nuclear receptors in cultured hypothalamic dopaminergic neurons.

By means of a monoclonal antibody against the rat liver L-triiodothyronine nuclear receptor and a polyclonal anti-tyrosine hydroxylase serum, it has been possible to demonstrate thyroid hormone nuclear receptors in immunoreactive tyrosine hydroxylase cell nuclei in fetal rat hypothalamic cultures. After 8 days in vitro, the ratio of tyrosine hydroxylase cells that were immunoreactive for the thyroid hormone receptor to those not stained for this receptor (64% to 36% respectively) remains unchanged despite an increase in the number of tyrosine hydroxylase-positive cells with time (from day 8 to day 21) in culture. The presence of thyroid hormone nuclear receptor in dopaminergic neurons is correlated with a morphological effect of L-triiodothyronine in this neuronal population. Our results demonstrate, for the first time, the presence of triiodothyronine nuclear receptors in fetal rat dopaminergic neurons and the existence of a cellular heterogeneity in the distribution of the thyroid hormone receptor. The presence of these receptors in fetal hypothalamic dopaminergic neurons suggests that some effects of L-triiodothyronine on the maturation of DA neurons may result from a direct effect of this hormone through an interaction with its specific nuclear receptors.

Animals↗
PubMed1989

Immunocytochemical localization of the nuclear 3,5,3'-triiodothyronine receptor in the adult rat: liver, kidney, heart, lung and spleen.

A monoclonal antibody was used for the localization of the nuclear T3 receptor in different tissues of the adult rat: the liver, kidney, heart, lung, spleen, testis, and pituitary. In the liver, the immunoreactivity was found uniformly distributed in the nuclei of hepatocytes. Sections incubated with a control ascitic fluid or with the same ascitic fluid pre-adsorbed with purified receptor showed no specific staining. In the kidney, the immunoreactivity was higher in the epithelial cell of the proximal convoluted tubes and juxtaglomerular cells. In the heart, only the myocardial cells were stained. In the lung, the immunoreactivity was confined to type II pneumocytes and alveolar macrophages. In the spleen, only a few mature lymphocyte and macrophage cell nuclei were stained. These results show that: 1) the abundance of the nuclear T3 correlates with previous studies using hormone binding techniques; 2) the nuclear T3 receptor is selectively located in certain cell types, which possess a precise local function.

Animals↗
PubMed1988

Influence of triiodothyronine (L-T3) on the morphological and biochemical development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium.

In cerebral hemisphere cultures initiated from 15-day-old rat embryos, the number of acetylcholinesterase-positive (AChE+) cells increased from 6.8 +/- 1.6 cells/well on day 3 to 112 +/- 16 cells/well on day 15. With time in culture, AChE+ cells increased both in size of the perikarya and neurite length. The addition of L-triiodothyronine (L-T3) at a concentration of 3 x 10(-8) M at the initiation of the culture had no effect on the number of AChE+ cells but significantly increased the size and neurite length of AChE+ neurons after 5 days in vitro. These morphological effects are associated with biochemical effects. L-T3 increased AChE activity in both a dose- and time-dependent manner (the stimulatory effect of L-T3 becomes significant between day 8 and day 15). Since a major part of AChE+ cells may be cholinergic neurons, we have also measured the effect of L-T3 on ChAT activity. L-T3 also increased ChAT activity in a dose and time dependent manner. Furthermore, treatment of cultures with L-T3 at different times in culture demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of L-T3 on ChAT activity is lost after 20 days in vitro. Studies of the time necessary for L-T3 to increase both ChAT and AChE activities show that 2 days and 15 days, respectively, are required for L-T3 to significantly stimulate both enzyme activities. This in vitro analysis demonstrated the morphological effect of L-T3 on the size and the neurite length of AChE+ cells. These effects are associated with biochemical effects on ChAT and AChE activities. Thus, it appears that thyroid hormones regulate several steps of neuronal maturation.

Acetylcholinesterase↗
PubMed1988

Useful parameters to predict the eventual mental outcome of hypothyroid children.

The Quebec Network for Genetic Medicine has followed the development of some 100 hypothyroid children treated by 1 month of age and evaluated at 18 months, 3 and 5 yr and the Griffiths Mental Development Scales, then at 7 and 9 yr with the Wechsler Intelligence Scale for Children Revised. Results show that the children as a group reach scores within the normal range of the tests. However, a few patients have low scores at each evaluation. Previously, we showed a correlation between a low serum thyroxine concentration, or a relatively retarded bone maturation before treatment, and low mental scores. To better characterize the significance of this relationship we correlated these pretreatment factors and the Wechsler Intelligence Scale for Children Revised results of 43 subjects reaching the age of 7 yr. Again, the same correlation was observed. Calculating a predictive factor (low thyroxine, less than 2 micrograms/dl and retarded bone surface, less than 0.05 cm2) from data recorded before therapy initiation, 10 of 13 children were correctly predicted to have I.Q. values less than 90. The use of these parameters might permit early intervention, and allow specific guidance of the more affected subjects.

Bone Development↗
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↗
PubMed1988

Effects of adenosine triphosphate and alkaline phosphatase on solubilized 3,5,3'-triiodothyronine-binding activity.

The T3-binding activity of salt-extractable nuclear proteins from rat liver was affected when ATP (2-10 mM; pH 8.0) was added concomitantly with T3 in the incubation medium. Scatchard analysis revealed that the equilibrium association constant was significantly reduced [5 mM ATP, 0.3 +/- 0.1 (+/- SE) 10(10) M-1; control, 1.1 +/- 0.15 X 10(10) M-1], but the maximum binding capacity remained unchanged. Similar values of inhibition were obtained when unbound receptors were preincubated with ATP. ATP achieved its maximal effect after 45 min of incubation at 30 C. Dilution experiments indicated that the effect of ATP was reversible. The inhibiting potency of nucleoside triphosphates at pH 8.0 was in the following order: ATP = CTP greater than GTP, whereas UTP had no effect. Nonhydrolyzable analogs of ATP were also inhibitory, and HPLC fractionation showed an approximately 98% recovery of ATP after incubation with nuclear extract. The adenine ring with at least two phosphates was essential, since ADP was as potent as ATP, whereas AMP had no effect. When the pH of the incubation medium was lowered to 7.3, the T3-binding activity was inhibited by ATP in the 0.1-1 mM range. Magnesium (3 mM) greatly increases the ATP effect at pH 7.3, but not at pH 8. The T3-binding activity was also drastically reduced when calf intestine alkaline phosphatase was added concomitantly in the incubation medium. Eight micrograms per ml enzyme were necessary to inhibit the T3 specific binding by 50% (30 C for 45 min). Scatchard analysis showed that the receptor affinity for T3 was decreased (control, 1.1 +/- 0.02 x 10(10) M-1; alkaline phosphatase, 0.41 +/- 0.03 x 10(10) M-1; n = 6), whereas the maximum binding capacity remained unchanged. Incubations performed with increasing concentrations of beta-mercaphoethanol (2.5, 5, 10, and 25 mM) revealed that the phosphatase inhibitory effect is thiol dependent. The inhibition was maximal at 2.5 mM and progressively decreased at 5 and 10 mM. No inhibition occurred at 25 mM. When a saturating concentration of T3 was employed, the specific binding was decreased at low thiol concentrations. These observations show that the nuclear T3 receptors may be modulated by ATP/ADP and phosphorylation/dephosphorylation processes. It is proposed that in vitro dephosphorylation leads to rapid oxydation of sulfhydryl groups which are essential for optimum T3 binding.

Adenosine Triphosphate↗
PubMed1988

Detection of the thyroxine-binding globulin (TBG) gene in six unrelated families with complete TBG deficiency.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Cloning, Molecular↗
PubMed1988

A study on the inheritance of thyroxine-binding globulin (TBG) deficiency from data obtained in 13 families detected by a neonatal screening program.

Thirteen families with thyroxine-binding globulin deficiency, detected through probands screened by the Quebec Network of Genetic Medicine, were investigated. These families were divided into two groups, depending on whether hemizygous males had low or undetectable serum thyroxine-binding globulin levels. Five families belonged to the low (hypo-TBGnemic) type while the remaining 8 families belonged to the absent (a-TBGnemic) type. On pedigree analysis, 10 families satisfied the requirements for an X-linked co-dominant mode of transmission. Three families, 2 a-TBGnemic and 1 hypo-TBGnemic, did not satisfy these requirements and three hypotheses had to be put forward to explain these situations: an autosomal recessive mode of transmission, a spontaneous mutation, or an extreme lyonisation of the defective X. Since the thyroxine-binding globulin gene has been localized to the X-chromosome, the two latter explanations appear to be the most plausible.

Female↗