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J Thibault

Publications and source records attributed to J Thibault.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5'-phosphate-dependent decarboxylase: a functional study.

The pyridoxal-5'-phosphate-dependent enzymes (B6 enzymes) are grouped into three main families named alpha, beta, and gamma. Proteins in the alpha and gamma families share the same fold and might be distantly related, while those in the beta family exhibit specific structural features. The rat aromatic L-amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal-5'-phosphate and belongs to the alpha family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B6 enzymes family, including the known three-dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site-directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild-type activity. Substitution of T246A resulted in 5% of the wild-type activity while the C311A mutant conserved 42% of the wild-type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies.

Amino Acid Sequence↗
PubMed1999

Lipid uptake across the wall of an expanded polytetrafluoroethylene vascular graft.

Previous studies have shown that vascular grafts were prone to inducing an atherosclerosis-like phenomenon, thus possibly jeopardizing their performance. Furthermore, lipid retention, observed in most synthetic arterial prostheses explanted from humans, appears to have an important role in the progression of this atherosclerotic process, therefore hindering the healing process and neo-intima formation of these synthetic conduits. The current study examined lipid concentration profiles across prosthesis membranes exposed to lipid dispersion under various transmural pressures, flow rates, and durations of exposure. It was demonstrated that the lipids rapidly permeated the prosthesis membrane, as lipid advection increased to a maximum, then steadily decreased until the membrane became completely impermeable to the fluid. The concentration of lipids within the grafts was monitored using FT-IR microspectroscopy, then correlated as a function of time in order to evaluate the mass transfer coefficients and lipid saturation concentration. Lipid sorption, as a function of time, was described by a mechanism taking into account two first-order kinetic models. The lipids were first rapidly adsorbed onto the Teflon(R), potentially influenced by the strong affinity of these lipids for the highly hydrophobic polytetrafluoroethylene polymer. This affinity then enhanced the germination of the lipid deposits that filled in the prosthesis wall. For lipid retention as a function of the transmural pressure and flow rate, no clear tendency was established.

Bioprosthesis↗
PubMed1999

Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections.

The olfactory bulb (OB) is involved in the processing of olfactory information particularly through the activation of its afferents. To localize their cell origin in sheep, a specific retrograde fluorescent tracer, Fluoro-Gold, was injected into the olfactory bulb of seven ewes. By using immunocytochemical techniques, retrogradely labeled neurons were colocalized with choline acetyltransferase, tyrosine hydroxylase, dopamine-beta-hydroxylase and serotonin to characterize cholinergic, noradrenergic and serotonergic Fluoro-Gold-labeled neurons. Most afferents originated from the ipsilateral side of the injection site. The OB received major inputs from the anterior olfactory nucleus (AON), the piriform cortex (PC), the olfactory tubercle, the diagonal band of Broca (DBB) and the amygdala. Other retrogradely labeled neurons were observed in the taenia tecta, the septum, the nucleus of the lateral olfactory tract, the preoptic area, the lateral hypothalamic area, the mediobasal hypothalamus, the lateral part of the premammillary nucleus, the paraventricular nucleus of the hypothalamus, the paraventricular thalamic nucleus, the central grey, the substantia nigra (SN), the ventral tegmental area (VTA), the lateral nucleus to the interpeduncular nucleus (IIP), the raphe and the locus coeruleus (LC). Contralateral labeling was also found in the AON, the PC, the SN compacta, the VTA, the IIP and the LC. Cholinergic Fluoro-Gold-labeled neurons belonged to the horizontal and vertical branch of the DBB. Noradrenergic afferents came from the LC and serotoninergic afferents came from the medial raphe nuclei and the 1IP. These data are discussed in relation with olfactory learning in the context of maternal behavior in sheep.

Animals↗
PubMed1999

Commercial polyurethanes: the potential influence of auxiliary chemicals on the biodegradation process.

This investigation elucidates some aspects of auxiliary chemicals on the biodegradation of two commercial polyurethanes (Pellethane and Corethane). The materials were incubated for 28 days with cholesterol esterase and/or with phosphatidylcholine. Extraction studies were carried out on the two materials, using different solvents, chosen on the basis of solvent polarity. FT-IR spectra for the extracted materials indicated the presence of poly(methylene)n oxide moities, silicone oil, bis-ethylene-stearamide, aromatic moities, and alkyd-urea compounds in Pellethane. Corethane materials were shown to contain some fatty acids, hydrocarbon waxes, ester-based species, and chlorinated compounds. Analysis of incubation solutions by high performance liquid chromatography failed to isolate methylene dianiline (MDA) or any of its derivatives from the various polymer incubation solutions. However, a methanol extract of Corethane samples that were incubated for 28 days in cholesterol esterase did show the presence of MDA. The absence of MDA in the Pellethane methanol extracted samples may reflect the differences in surface additives found for this material versus the Corethane. FT-IR/ATR analysis of polymer surfaces exposed to cholesterol esterase/phospholipids mixture showed that there was an increase in the uptake of phospholipids over samples that were incubated in phospholipid dispersion alone. The results of this study show that some of the auxiliary chemicals found in commercial polyurethanes may hinder the specific release of hydrolytic degradation products and delay polymer degradation. However, it should be recognized that the surface layer containing these compounds is susceptible to change following the interaction between the polyurethane-based devices and elements of the host environment (i.e. lipids, enzymes, etc.). Hence, recognition and identification of these changes will ultimately be important in assessing a commercial polymer's blood compatibility characteristics.

Biodegradation, Environmental↗
PubMed1999

[Response of vasopressin and tyrosine hydroxylase expressing neurons of the rat supraoptic nucleus to chronic osmotic stimulation].

This study has evaluated the dynamic of intracellular vasopressin and tyrosine hydroxylase contents in the neuron cell bodies in the supraoptic nucleus and in the axons of the posterior lobe in rats drinking 2% NaCl for 1, 2, and 3 weeks. The number of vasopressin-immunoreactive neurons increased by the end of the second week of osmotic stimulation that might be explained by the onset of vasopressin synthesis in the neurons which do not synthesize this neurohormone under normal physiological conditions. The concentration of vasopressin fell down continuously during the first two weeks of salt-loading, apparently, due to predominance of the vasopressin release over its synthesis. Over the third week of salt-loading, the intracellular concentration of vasopressin was not changed significantly suggesting the establishment of the dynamic equilibrium between the vasopressin synthesis and release. The number of tyrosine hydroxylase-immunoreactive neurons and the amount of tyrosine hydroxylase in cell bodies and the large axonal swellings, Herring bodies, increased gradually showing that the rate of tyrosine hydroxylase synthesis prevailed over that of its enzymatic degradation. Thus, the chronic stimulation of vasopressin neurons is accompanied by a number of the adaptive reactions; the most important is related to the onset of vasopressin and tyrosine hydroxylase synthesis in the neurons which do not synthetize both of them under normal conditions.

Animals↗
PubMed1998

Immunolocalization of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, dopamine, and serotonin in the forebrain of Ambystoma mexicanum.

To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.

Ambystoma↗
PubMed1998

Lipid uptake in expanded polytetrafluoroethylene vascular grafts.

PURPOSE: The mechanisms of vascular prosthesis failure are reported to be associated, in part, with an atherosclerotic degenerative process that is related to an abnormal lipid infiltration. The lipid uptake in expanded polytetrafluoroethylene (ePTFE) vascular grafts was reproduced in vitro, and the effect of time on the permeability of these prostheses was studied. METHODS: Water permeability tests were carried out under dynamic flow conditions at various hydrostatic pressures. Lipid uptake was simulated by circulating a phosphatidylcholine suspension inside an expanded Teflon prosthesis under pulsatile or continuous transmural pressure ranging between 80 mm Hg and 180 mm Hg, at a flow rate of 500 mL/min and 2000 mL/min, for a duration ranging from 2 hours to 1 month. RESULTS: Water permeability tests indicated that under hydrostatic pressures of 180 mm Hg and 300 mm Hg, water percolated through the prosthesis wall after an exposure of 720 minutes and 75 minutes, respectively. After exposing the prostheses to the lipid dispersion under the various flow conditions, the fluid convection through the wall occurred. Preferential convection pathways with a constant periodicity were observed across the length of each prosthesis and were, therefore, associated with regularly spaced perforations depicted in the structure of the devices. Phospholipids gradually agglomerated within the prosthesis wall, allowing a restrictive molecular mobility. Infrared spectroscopy results indicated that the lipid uptake depended on the transmural pressure and time of exposure. CONCLUSION: The occurrence of the membrane permeability may be associated with the dilatation and plastic deformation of the prosthesis. Lipid uptake occurs in ePTFE grafts after an aggressive kinetic process.

Blood Vessel Prosthesis↗
PubMed1998

Presence of galanin in dopaminergic neurons of the sheep infundibular nucleus: a double staining immunohistochemical study.

The distribution of tyrosine hydroxylase (TH) and of galanin immunoreactive (IR) neurons were examined in the sheep infundibular nucleus. Antisera raised against TH and galanin were used on adjacent sections and for double immunohistochemical staining of the same sections. There was considerable overlap in the distribution of TH and galanin-IR neurons in the medial part of the nucleus. Most of the galanin-IR neurons were also TH-IR, but less than 50% of the TH-IR neurons also expressed galanin immunoreactivity. Neurons immunoreactive to TH alone were observed close to the third ventricle and in the rostral part of the infundibular nucleus. In the median eminence, TH and galanin-IR fibres overlapped mainly in the lateral and dorsal parts of the external layer, but the colocalisation of both antigens could not be assessed on the available material. Thus, in sheep, the population of catecholaminergic neurons of the infundibular nucleus may be subdivided into different subpopulations according to their peptide content, but does not appear segregated as in rat and human.

Animals↗
PubMed1998

A continuous and pulsatile flow circulation system for evaluation of cardiovascular devices.

The design of a nonpulsatile and pulsatile system using a centrifugal pump is presented. To induce a pulsatile flow with a centrifugal pump, an independent pneumatically driven unit provided flow patterns over a wide range of frequencies and amplitudes. The pulsatile flow was generated by the axial displacement of a cylinder that periodically compressed the flexible conduit that is connected to the pump. The system can accommodate flow rates up to 6,000 ml/min and transmural pressures up to 500 mm Hg and is capable of maintaining the pressure at a constant value. This circuit produced reproducible pressure waves having a frequency up to 4 Hz. The periodicity of the transmural pressure between 80 and 180 mm Hg was similar to the pressure wave propagation observed in peripheral circulation. Capable of adequately reproducing continuous and pulsatile flow, the apparatus is therefore versatile to allow in vitro evaluation of cardiovascular devices.

Centrifugation↗
PubMed1997

Impact of Rantes and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase.

RANTES and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells constitute the MCP-1 or C-X-C class. The roles of most of these chemokines are not well known, although members of the C-X-C family are inflammatory agents. Here, we report that intradermal injection of RANTES 10 ng/50 microL subcutaneously in the abdominal skin produced a strong inflammatory reaction, as evidenced by Evans blue dye, greater than FMLP (10(-6) mol/L) (approximately 57%); while MCP-1, 10 ng/50 microL was less effective than FMLP (10(-6) mol/L) (approximately 54%). Moreover, the histologic analysis of the cells stained with Toluidine blue (0.1%) were analyzed at a magnification of x40). RANTES 10 ng/50 microL and LPS produced higher numbers (142 +/- 11 and 193 +/- 21 of cells/200 mm2, respectively) of basophilic cell accumulation in the skin injection sites compared with FMLP (10(-6) mol/L) (127 +/- 14/200 mm2), while MCP-1 10 ng/50 microL was less effective (88 +/- 10/200 mm2). Electron microscopy (x13,800) studies of skin injection sites revealed that RANTES was chemoattractant for mast cells. In a Northern blot analysis from homogeneous tissue biopsy from the intradermal injection sites, RANTES was more potent than MCP-1 in increasing histidine decarboxylase (HDC) mRNA, the sole enzyme responsible for the production of histamine from histidine. Since PGD2 is formed by mast cells on cell activation, we also studied the effect of RANTES and MCP-1 on PGD2 production in inflamed tissue in vivo. RANTES (20, 10, and 5 ng) and MCP-1 (20, 10, and 5 ng) strongly stimulated PGD2, in a dose-dependent manner, with a potency rank order of RANTES (10 ng/mL) approximately two times greater than MCP-1 (10 ng/mL).

Animals↗
PubMed1997

Long-term monitoring of extracellular dopamine concentration in the rat striatum by a repeated microdialysis procedure.

This study examined a protocol for repeated measurement of the extracellular dopamine (DA) concentration in the rat striatum by microdialysis. Rats were implanted with a guide cannula in the striatum and the probe was inserted on each dialysing day, i.e. ten times over a 23 day period. During this period the animals were submitted to a control saline treatment. DA concentration was measured using the no-net-flux method. In these conditions, DA concentration remained remarkably constant over the 23 day period. The histological analysis using glial fibrillary acidic protein (GFAP), dopamine (DA) and tyrosine hydroxylase (TH) immunocytochemistry showed a moderate gliosis and a discrete increase of immunoreactivity of catecholaminergic fibres around the probe implantation site. This increase is probably related to a plasticity of the dopaminergic system in response to the lesion due to the probe implantations. This study shows that such a paradigm makes possible to measure the whole time course of the DA concentration in the rat striatum during chronic treatments with psychoactive drugs such cocaine or other compounds acting in the nigrostriatal system.

Animals↗
PubMed1997

RANTES is a pro-inflammatory chemokine and chemoattracts basophil cells to extravascular sites.

RANTES (regulated upon activation normal T expressed and secreted) is another member of the intercrine beta subfamily which acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. In this work, the effect of RANTES was studied on rat skin injection sites. Rats were intradermally injected with 50 microliters of RANTES, at different concentrations, fMet-Leu-Phe (FMLP), or LPS (positive controls) or PBS vehicle (negative control). The animals were then injected with 0.6 ml of Evans' blue in the tail vein in order to obtain a blue colour in the areas where the compounds were injected. After 4 h the rats were killed and the maximum diameter of the blue extravasation area was measured. The coloured areas were then excised and optical and electron microscopic studies were performed. In addition, in some of the excised tissue, a Northern blot analysis for histidine decarboxylase (HDC) mRNA was performed along with an estimation of the amount of histamine generated in the tissue injection sites. In these studies it was found that intradermal injections of 5, 2.5, and 1.25 x 10(-5) M RANTES produced a strong inflammatory response with the accumulation of a great number of basophil cells compared with the PBS (50 microliters) negative control, or FMLP (10(-6) M/50 microliters) or LPS (10 ng/50 microliters) positive control, after 4 h. Moreover, 5, 2.5, 1.25 x 10(-5) M RANTES produced a dose-response stimulation of HDC mRNA in the tissues of skin injection sites. The increasing number of basophils in the RANTES inflamed tissues led to augmentation of histamine content, compared with the PBS control. In conclusion, the pro-inflammatory chemokine RANTES stimulates the generation of HDC mRNA in skin injection sites.

Animals↗
PubMed1997

Expression, purification, and characterization of rat aromatic L-amino acid decarboxylase in Escherichia coli.

A cDNA encoding rat aromatic L-amino acid decarboxylase (AADC) was successfully expressed in Escherichia coli using a T7 RNA polymerase expression system. Two types of expression vectors were tested and revealed to be equivalent to produce AADC. The enzyme was purified in both cases. The ratio of recovery of the pure active recombinant protein was better when the purification of the protein was made easier by addition of a short His-Tag at the C-terminal moiety of AADC, as achieved in the case of pET-20b+ vector expression. Spectral characteristics of the bound pyridoxal-5'-phosphate were essentially identical to the spectral properties of rat AADC. Kinetic constants Km and Vmax of recombinant AADC for the natural substrates L-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mM and 8444 U/mg, and 0.066 mM and 1813 U/mg, respectively. These values were in good agreement with previously reported values for AADC of the rat and other mammalian species.

5-Hydroxytryptophan↗
PubMed1997

Long-lasting effect of catecholamine deficiency on differentiating vasopressin and oxytocin neurons in the rat supraoptic nucleus.

According to our earlier study, the catecholamine depletion in neonatal rats resulted in stimulation of the vasopressin and oxytocin gene expression in the neurons of the supraoptic nucleus. The present study extends this line, evaluating whether the catecholamine deficiency provides a long-lasting effect on the differentiating vasopressin and oxytocin neurons of the supraoptic nucleus. Catecholamines were depleted by daily injections of an inhibitor of the catecholamine synthesis, alpha-methyl-p-tyrosine, first, to pregnant rats from the 9th to the 21st day of gestation and, then, to their pups from the 2nd to the 10th postnatal day. The animals, injected with saline instead of drugs, served as controls. The pharmacologically-treated and control rats were kept for four months under normal laboratory conditions until processing the materials for semi-quantitative in situ hybridization and immunocytochemistry of vasopressin and oxytocin messenger RNAs and peptides, respectively. There were no differences in the vasopressin and oxytocin messenger RNA concentrations in the supraoptic nucleus in rats following preliminary catecholamine depletion compared to controls. Conversely, the catecholamine deficiency resulted in an increased content of the vasopressin-immunoreactive material in cell bodies and processes. This was also the case for the oxytocin-immunoreactive cell bodies but only in females, suggesting an interference of catecholamines with sexual steroids in their action. The number and size of vasopressin and oxytocin neurons did not change in pharmacologically-treated rats compared to the controls. Thus, the catecholamine deficiency in the course of the neuron differentiation resulted in a long-lasting augmentation of the intracellular content of vasopressin and oxytocin but did not influence the vasopressin and oxytocin gene expression. This might be explained rather by the reduced level of peptide release than by an increased level of the peptide production.

Animals↗
PubMed1997

Comparison of glutamate and N-methyl-D-aspartate toxicities on rat mesencephalic primary cell cultures.

Excitotoxicities of glutamate and NMDA were studied on primary cultures of rat embryonic substantia nigra. The toxicity of the general neuronal population (identified with neuron specific enolase-NSE) was compared with that of dopaminergic neurons (identified with TH antibodies). We have shown that there exists a time-dependent toxicity to glutamate in 9 d old cultures in vitro and exposures as short as 5 min are significantly toxic. By comparing the effects of long time exposures (24 h) to NMDA and glutamate, we can show dose-dependent toxicity; however NMDA shows a less marked effect, especially at high doses (> 500-1000 microM) as opposed to less potent lower doses (< 500 microM). In comparison to the general population of NSE-positive mesencephalic neurons, TH-positive neurons seem to exhibit a similar vulnerability to EAA. The fact that TH-positive neurons are only partially protected against glutamate toxicity by the non-competitive NMDA antagonist TCP indicates that they are more susceptible to non-NMDA mediated neurotoxicity than the general neuronal population.

Animals↗
PubMed1997

Active rat aromatic-L-amino acid decarboxylase as a fusion protein in Escherichia coli.

The DNA sequence encoding rat aromatic-L-amino acid decarboxylase (AADC) was inserted into the Escherichia coli (E. coli) expression vector pMAL-c2. This clone produced a fusion protein able to catalyze the conversion of L-DOPA to dopamine. After purification and treatment of the fusion protein by factor Xa (FXa), an enzymatically active form of the enzyme resistant to FXa was isolated. It showed kinetic constants, Vmax, K(m) and enzymatic properties very similar to those obtained previously for the mammalian enzyme. This method for obtaining active AADC appears to be useful for initiating the study of the catalytic activity of this protein because it permitted the rapid isolation and the stabilization of an active form of the enzyme.

Animals↗
PubMed1997

Isolation and characterization of ovine tyrosine hydroxylase mRNA.

Tyrosine hydroxylase (TH) cDNA has been characterized in rodents and primates, but only a few studies have been developed in ungulates, except in cows. Because sheep is a species used for many physiological studies, it was of interest to clone TH cDNA in this species. Ovine TH cDNA was purified from a library of sheep adrenal glands. The entire cDNA was 1,721 bp long. It presented a higher percentage of similarity with bovine TH cDNA (93%) than with rodent cDNAs (75%). The deduced amino acid sequence was 490 amino acids long and had 96% similarity with the bovine amino acid sequence. The entire cDNA and different fragments obtained with endonuclease restriction enzymes were cloned in plasmid pUC 18 and were labeled with 35S-dATP to detect TH mRNA by in situ hybridization. Strong labelings were observed on adrenal medulla and on noradrenergic and dopaminergic neurons in the sheep but also in the cow and pig. This labeling matched completely TH immunohistochemical staining obtained on the same sections with anti-TH antibodies. Ovine TH cDNA is a useful tool to study the variations of TH mRNA levels in sheep catecholaminergic neurons.

Amino Acid Sequence↗