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J Thibault

Publications and source records attributed to J Thibault.

15 recordsLinked to original sources
PubMed1992

Characterization and partial purification from pheochromocytoma cells of an endogenous equivalent of scyllatoxin, a scorpion toxin which blocks small conductance Ca(2+)-activated K+ channels.

This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca(2+)-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.

Animals↗
PubMed1992

Immunohistochemical colocalization of tyrosine hydroxylase and estradiol receptors in the sheep arcuate nucleus.

In sheep, the arcuate nucleus contains numerous tyrosine hydroxylase (TH) and estradiol receptor (rE2) immunoreactive (IR) perikarya and it has been shown previously in this species that catecholaminergic neurons can mediate the gonadal steroid action on the reproductive function. In the present study, double immunohistochemical labelling with antibodies against TH and rE2 have been used to demonstrate the presence of rE2 in TH-IR neurons in the arcuate nucleus where the distribution of TH-IR and rE2-IR neurons overlap each other. Only less than 10% of all the rE2-IR perikarya presented TH immunoreactivity. It was therefore hypothesized that either such a low number of double labelled neurons can support the effects of estradiol in this area or that the effect of this steroid was indirect. In the latter case it might be first mediated by beta-endorphin neurons which have been previously described in this nucleus.

Animals↗
PubMed1992

Mapping of the dopa decarboxylase gene to the 11A band of the murine genome.

A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.

Animals↗
PubMed1992

Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex. Reversal of the endogenous feedback inhibition by phosphorylation of serine-40.

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.

Adrenal Gland Neoplasms↗
PubMed1992

Preparation of a rat brain histidine decarboxylase (HDC) cDNA probe by PCR and assignment of the human HDC gene to chromosome 15.

The formation of histamine from its precursor histidine is catalyzed by histidine decarboxylase (HDC), a pyridoxal phosphate (PLP)-dependent decarboxylase. The knowledge of sequence similarities between various rodent HDCs permitted us to prepare a rat brain HDC cDNA probe. After reverse transcription of rat brain polyA + mRNA, the HDC cDNA obtained was amplified by the polymerase chain reaction using two specific primers. The resulting 1019-bp DNA was cloned in the p-MAL vector. Its sequence corresponds to the published data on rat fetal liver HDC. This 1019-bp rat probe detected two BamHI sequences in man; these were assigned to chromosome 15 by somatic hybrid cell analysis. According to the well-known homology between human chromosome 15 and mouse chromosome 2, the result obtained is in agreement with the published localization of HDC on mouse chromosome 2. The mapping of the human HDC gene on chromosome 15 is an original contribution to the chromosomal assignment of related PLP-dependent decarboxylases.

Animals↗
PubMed1992

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt, Triturus alpestris (Urodela).

Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA- and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, originating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.

Animals↗
PubMed1991

Dopamine increases the expression of tyrosine hydroxylase and aromatic amino acid decarboxylase in primary cultures of fetal neurons.

Previous studies, aimed at identifying which diffusible signals may influence the differentiation of embryonic neurons towards the monoaminergic phenotypes during brain development, have shown that serotonin itself could promote the 'serotoninergic-like properties' of hypothalamic cells from mouse embryos. We presently reinvestigated such 'autocrine/paracrine' regulatory mechanisms by exposing dissociated cell cultures from embryonic rat hypothalamus and brain stem to dopamine--or related agonists--in an attempt to influence their differentiation towards the catecholaminergic phenotype. Chronic treatment of cells by dopamine or apomorphine (a mixed D1/D2 agonist), but not selective D1 and D2 agonists, significantly increased the number of cells that expressed tyrosine hydroxylase (TH: as assessed with a specific anti-TH antiserum) and the activity of aromatic L-amino acid decarboxylase (AADC) in the cultures. Furthermore, apomorphine treatment also decreased the levels of cholecystokinin-like material in primary cultures from the brainstem (but not the hypothalamus) where both dopamine and cholecystokinin are--partly--colocalized in mesencephalic dopaminergic neurons. The maximal effects of both dopamine and apomorphine on TH expression and AADC activity occurred earlier in the brainstem (on cells from 14- to 15-day-old embryos) than in the hypothalamus (on cells from 15- to 16-day-old embryos), in line with the well established caudo-rostral maturation of the rat brain. Furthermore both the expression and the dopamine-induced modulation of AADC activity and TH immunoreactivity appeared to occur independently of each other. Present and previous data are in agreement with a possible autocrine/paracrine action of dopamine and serotonin during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Quantitative image analysis with densitometry for immunohistochemistry and autoradiography of receptor binding sites--methodological considerations.

Major technical progress in the development of computer-based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion-coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections. Immunohistochemistry after revelation by a color-based enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment of a scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail. Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of alpha-methyltyrosine (AMPT), a well-known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D1 and D2 receptor sites, particularly the dual patch-matrix compartments.

Animals↗
PubMed1991

Quantitative autoradiography of tyrosine hydroxylase immunoreactivity in the rat brain.

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.

Animals↗
PubMed1990

Preparation of a human DOPA decarboxylase cDNA probe by PCR and its assignment to chromosome 7.

The reverse transcription of mRNA of a human pheochromocytoma using an oligonucleotide complementary to rat DOPA decarboxylase (DDC) sequence as a primer, gave rise to a single strand cDNA. This resultant cDNA permitted by PCR amplification the preparation and cloning in PUC 19 of a human DDC probe of 747 base pairs. The choice of primer was dictated by the presence of tryptophan codons in the DOPA decarboxylase sequence which were conserved in different species throughout evolution. The presence of mismatches on the primers was not an obstacle to a specific amplification and the probe sequence was found identical to the human DDC cDNA sequence. This probe, labelled by nick translation detected on Southern blot of human-rodent hybrids, bands on human DNA after EcoRI, BamHI or HindIII digestion. The results with EcoRI were explicit and drove to the conclusion that DDC is located on chromosome 7. Five bands were obtained on human DNA digested with EcoRI, indicating that either numerous introns could interrupt the coding sequence of DDC gene or duplicated sequences could be present in chromosome 7.

Base Sequence↗
PubMed1979

Immunization of adult rats against 2.5 S NGF: effects on the peripheral sympathetic nervous system.

The biochemical and morphological changes effected by immunization of adult rats with 2.5 S mouse nerve growth factor (NGF) were studied in sympathetic ganglia and in representative target organs. This immunization procedure maintains high levels of circulating anti NGF-antibody for periods of months. Morphological analysis revealed a general reduction in the size of the adrenergic neurons in the superior cervical ganglion (SCG) which was also reflected at the biochemical level by a 30% decrease in total protein content and a 50--60% reduction in the total activities of all norepinephrine-synthesizing enzymes. However, there was no change in total choline acetyltransferase activity. The biochemical and morphological changes observed in the SCG seem to be confined to the neuronal cell body, since at any stage of immunization target organs (the submandibular and the pineal gland) remained unaffected. All sympathetic ganglia investigated--except the superior mesenteric ganglion--responded in a similar way to the immunization against 2.5 S NGF. These changes in the adrenergic cell bodies were largely reversible. The recovery of normal enzyme activities followed closely the decrease of the antibody titer after cessation of immunization boosting. This indicates that cell death is not caused by anti NGF-antibodies in ganglia of adult animals. Thus, in contrast to adrenergic neurons from newborn animals, which depend on NGF or a crossreacting NGF-like material for survival, differentiated adrenergic neurons need this factor for the maintenance of their normal function but not for survival.

Animals↗