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Biomedical subjects

P Ravassard

Publications and source records attributed to P Ravassard.

18 recordsLinked to original sources
PubMed2006

Sleep dynamics: a self-organized critical system.

In psychiatric and neurological diseases, sleep is often perturbed. Moreover, recent works on humans and animals tend to show that sleep plays a strong role in memory processes. Reciprocally, sleep dynamics following a learning task is modified [Hubert, Nature (London) 02663, 1 (2004), Peigneux, Neuron 44, 535 (2004)]. However, sleep analysis in humans and animals is often limited to the total sleep and wake duration quantification. These two parameters are not fully able to characterize the sleep dynamics. In mammals sleep presents a complex organization with an alternation of slow wave sleep (SWS) and paradoxical sleep (PS) episodes. Moreover, it has been shown recently that these sleep episodes are frequently interrupted by micro-arousal (without awakening). We present here a detailed analysis of the basal sleep properties emerging from the mechanisms underlying the vigilance states alternation in an animal model. These properties present a self-organized critical system signature and reveal the existence of two W, two SWS, and a PS structure exhibiting a criticality as met in sand piles. We propose a theoretical model of the sleep dynamics based on several interacting neuronal populations. This new model of sleep dynamics presents the same properties as experimentally observed, and explains the variability of the collected data. This experimental and theoretical study suggests that sleep dynamics shares several common features with critical systems.

Journal Article↗
PubMed2006

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway.

The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

Animals↗
PubMed2005

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells.

AIMS/HYPOTHESIS: Gene transfer into pancreatic beta cells, which produce and secrete insulin, is a promising strategy to protect such cells against autoimmune destruction and also to generate beta cells in mass, thereby providing a novel therapeutic approach to treat diabetic patients. Until recently, exogenous DNA has been directly transferred into mature beta cells with various levels of success. We investigated whether exogenous DNA could be stably transferred into pancreatic stem/progenitor cells, which would subsequently differentiate into mature beta cells expressing the transgene. METHODS: We designed transplantation and tissue culture procedures to obtain ex vivo models of pancreatic development. We next constructed recombinant lentiviruses expressing enhanced green fluorescent protein (eGFP) under the control of either the rat insulin promoter or a ubiquitous promoter, and performed viral infection of rat embryonic pancreatic tissue. RESULTS: Embryonic pancreas infected with recombinant lentiviruses resulted in endocrine cell differentiation and restricted cell type expression of the transgene according to the specificity of the promoter used in the viral construct. We next demonstrated that the efficiency of infection could be further improved upon infection of embryonic pancreatic epithelia, followed by their in vitro culture, using conditions that favour endocrine cell differentiation. Under these conditions, endocrine stem/progenitor cells expressing neurogenin 3 are efficiently transduced by recombinant lentiviral vectors. Moreover, when eGFP was placed under the control of the insulin promoter, 70.4% of the developed beta cells were eGFP-expressing cells. All of the eGFP-positive cells were insulin-producing cells. CONCLUSIONS/INTERPRETATION: We have demonstrated that mature rat pancreatic beta cells can be stably modified by infecting pancreatic stem/progenitor cells that undergo endocrine differentiation.

Animals↗
PubMed2004

Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain.

We have recently identified a third subtype of glutamate vesicular transporter (VGLUT) named VGLUT3. In the present study, we provide a detailed account of the regional and cellular distributions of VGLUT3 in the rat brain, using specific nucleotide probes and antisera. The distribution of VGLUT3 protein was compared with that of the other vesicular transporters (VGLUT1 and VGLUT2). All the areas expressing VGLUT3 also contain high levels of VGLUT1 and -2 proteins, but, at a finer level of analysis, the distribution of the three subtypes differs. Unlike VGLUT1 and -2, VGLUT3 expression is limited to discrete cell populations. Neurons containing VGLUT3 transcript are essentially observed in the caudate-putamen, the olfactory tubercle, the nucleus accumbens, the hippocampus, the interpeduncular nucleus and the dorsal and medial raphe nuclei. More scattered populations of VGLUT3 expressing neurons are found in the cerebral cortex. The distribution of the VGLUT3 protein, as determined with specific antisera, overlaps with that of the transcript in the caudate-putamen, olfactory tubercles, hippocampus, cortex, interpeduncular nucleus, and raphe nuclei, suggesting that VGLUT3 is essentially present in local projection neurons in these regions. Microscopic examination reveals staining of terminals and perikarya. Furthermore, co-localization studies indicate that VGLUT3 is present in GABAergic interneurons in the hippocampus, as well as in the interpeduncular nucleus. However, other regions, such as the substantia nigra (pars compacta), the ventral tegmental area, and the parabigeminal nucleus, receive a dense VGLUT3 terminal labeling although they do not contain VGLUT3 expressing neurons. In these regions, VGLUT3 immunoreactivity may be present in terminals of long projecting neurons. This subclass of glutamatergic afferents differs from other "classical" excitatory terminals that express VGLUT1 or VGLUT2. The distribution of VGLUT3 in the rat brain suggests an unsuspected function of vesicular glutamate transport in subsets of interneurons and in neuromodulatory neurons.

Amino Acid Transport Systems, Acidic↗
PubMed2001

The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons.

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Amino Acid Transport Systems↗
PubMed2001

CREM and ICER are differentially implicated in trans-synaptic induction of tyrosine hydroxylase gene expression in adrenal medulla and sympathetic ganglia of rat.

Reserpine treatment leads to a trans-synaptic increase of the tyrosine hydroxylase (TH) gene transcription rate, mRNA and protein levels in catecholaminergic tissues including the adrenal medulla (AM) and the superior cervical ganglia (SCG). The TPA-responsive element plays an important role in the trans-synaptically-induced transcription of the TH gene in the AM, whereas it does not appear to be involved in the SCG (Trocmé et al. [1997] J. Neurosci. Res. 48:489-498). In this study, we show that another regulatory sequence of the TH proximal promoter, the cAMP-responsive element (CRE), binds different factors in the AM and in the SCG. To elucidate the dynamics of promoter regulation a complete time course analysis was conducted. Reserpine treatment enhances, between 1 hr and 8 hr after the injection, the expression and the binding of the repressor ICER in the AM, whereas in the SCG it enhances the binding of CREM factors. These results suggest that the mechanisms mediating trans-synaptic induction of the TH gene are different in the AM and SCG. The interplay between positive and negative transcription factors and their kinetics of action are responsive of the long-term regulation of the TH gene.

Adrenal Medulla↗
PubMed2001

Identification and characterization of a lysosomal transporter for small neutral amino acids.

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Alanine↗
PubMed1999

ZNF74, a gene deleted in DiGeorge syndrome, is expressed in human neural crest-derived tissues and foregut endoderm epithelia.

DiGeorge syndrome (DGS) is a developmental disorder associated with large hemizygous deletions on chromosome 22q11.2. ZNF74 zinc finger gene is a candidate from the commonly deleted region. To address the potential involvement of ZNF74 in DGS, its human developmental expression pattern has been assessed. In situ hybridization on Carnegie Stage 18 embryos revealed that ZNF74 expression is limited to specific neural crest-derived tissues and neuroepithelium of the spinal cord as well as to foregut endoderm epithelia (esophagus and respiratory tract). Interestingly, ZNF74 expression was detected in the wall of the pulmonary artery and aorta and in the aortic valve, which are populated by neural crest-derived cells. This finding is significant, considering that DGS is believed to result from defective neural crest contributions and that outflow tract and aorticopulmonary septation defects are typical features of the DGS phenotype. Thus, the restricted expression of ZNF74 in structures affected in DGS suggests a role for this putative regulator of gene expression in aspects of the DGS phenotype.

Aorta↗
PubMed1999

A human tyrosine hydroxylase isoform associated with progressive supranuclear palsy shows altered enzymatic activity.

A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generated by alternative splicing and characterized by the absence of the third exon was recently identified. The corresponding putative protein lacks 74 amino acids including Ser31 and Ser40, two major phosphorylation sites implicated in the regulation of HTH activity. These mRNA species are detected in adrenal medulla and are overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia. In the present work, an HTH protein isoform lacking exon 3 was identified in human adrenal medulla. For this purpose, an antibody was raised against the HTH exon 3. The effect of the removal of exon 3 on the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTHDelta3) to the equivalent protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTHDelta3 has 30% of the maximal velocity of GST-HTH3. Moreover, the skipping of exon 3 results in the absence of activation of GST-HTH by heparin and increases by 10-fold the retroinhibition constant for dopamine, demonstrating the involvement of exon 3 in the regulation of HTH enzymatic activity. The identification of a variably expressed HTH isoform that lacks an exon implicated in activity regulation supports the view that HTH alternative splicing contributes to the functional diversity within the catecholaminergic system and may be implicated in some neurological diseases.

Adrenal Glands↗
PubMed1998

Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation.

Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1998

A tetranucleotide polymorphic microsatellite, located in the first intron of the tyrosine hydroxylase gene, acts as a transcription regulatory element in vitro.

The polymorphic HUMTH01 microsatellite, located in the first intron of the tyrosine hydroxylase gene is characterized by a tetranucleotide core motif. The 10 repeat allele of this microsatellite exhibits two sequence variants: an imperfect repeat and a perfect repeat. Here we present evidence that this tetrarepeat is endowed with regulatory properties. Constructions were made linking the 10 repetition alleles to the luciferase reporter gene under the control of a thymidine kinase minimal promoter. In transient transfection experiments in HeLa, PC12 and SK-NSH cell lines these repeated sequences increased the basal transcription up to 9-fold. This effect was independent of the sequence orientation, a feature characteristic of an enhancer element. In electrophoretic mobility shift assays these tetrameric repeated sequences form specific complexes with HeLa cell nuclear extracts. Competition experiments with heterologous sequences suggest that proteins of the Fos-Jun family may be involved in the formation of these complexes, although other unidentified transacting factors bind to these sequences. These results thus implicate the HUMTH01 microsatellite in the regulation of tyrosine hydroxylase gene expression. Tetrarepeated sequences of this type may constitute a new class of regulatory elements.

Alleles↗
PubMed1997

Relax promotes ectopic neuronal differentiation in Xenopus embryos.

We previously isolated a novel rat cDNA encoding a basic helix-loop-helix transcription factor named Relax, whose expression in the developing central nervous system is strictly limited to discrete domains containing precursor cells. The timing of Relax expression coincides with neuronal differentiation. To investigate the involvement of Relax in neurogenesis we tested whether Relax activated neural genes in the ectoderm by injecting Relax RNA into Xenopus embryos. We demonstrate that ectopic Relax expression induces a persistent enlargement of the neural plate and converts presumptive epidermal cells into neurons. This indicates that Relax, when overexpressed in Xenopus embryos, has a neuronal fate-determination function. Analyses both of Relax overexpression in the frog and of the distribution of Relax in the rat neural tube strongly suggest that Relax is a neuronal fate-determination gene.

Animals↗
PubMed1997

An ADP-ribosylation-factor(ARF)-like protein involved in regulated secretion.

A rat ADP-ribosylation factor(ARF)-like protein named ARL184 was identified by cDNA cloning. The corresponding recombinant protein had an apparent molecular mass of 22,000. The deduced amino acid sequence had 55% identity with the human ARL1 and four functional GTP-binding sites. Immunofluorescent confocal microscopy studies showed that ARL184 was present in the cytosol as well as in the Golgi apparatus, raising the possibility that it has a role in a secretory pathway. The involvement of this ARF-like protein in secretion was confirmed by demonstrating that ARL184 potentiated acetylcholine release in stably transfected PC12 cells. Collectively these results suggest that this ARL protein is a component of a regulated secretory pathway involved in Ca2(+)-dependent release of acetylcholine.

ADP-Ribosylation Factors↗
PubMed1997

Relax, a novel rat bHLH transcriptional regulator transiently expressed in the ventricular proliferating zone of the developing central nervous system.

A number of basic helix-loop-helix containing proteins have been shown to be required for neural development at different sites or times. Here, Relax, a novel rat basic helix-loop-helix transcriptional regulator, has been isolated and characterized. Analysis of the temporal and spatial distributions shows that the Relax transcripts are detected exclusively in the central nervous system, in discrete regions from embryonic day 11.5 to 18.5. Most strikingly, Relax is expressed along two major boundaries that define the longitudinal axis in the spinal cord and the hindbrain and is a marker of the anterior tip of this axis in the forebrain. Relax-expressing cells are strictly localized in the ventricular zone of the neural tube, where neural progenitors originate. This unique pattern of expression suggests that Relax is involved in neural fate determination.

Animals↗
PubMed1997

Detection of two new polymorphic sites in the human interleukin-1 beta gene: lack of association with schizophrenia in a French population.

The pathogenesis of schizophrenia might involve abnormal development of the human brain. Interleukin-1 beta is a cytokine implicated in the development of the central nervous system and therefore its gene is a candidate gene in schizophrenia. Polymorphisms within the coding sequence and the 3'UTR of the IL1 beta gene were searched for using PCR-SSCP. Two polymorphisms, 1B-175/1B-173 and 1B-1765/1B-1763 were found in addition to the previously published TaqI site. Furthermore, a mutant was found in codon 106 (exon 5) of the IL1 beta gene located next to the published polymorphism at the TaqI site and abolishing this site. This novel mutation encodes an Asp in place of an Asn and was only observed in one patient in our French population. Association studies were conducted with the polymorphisms 1B-175/1B-173 and TaqI. There was no allelic or genotypic association between either of the two polymorphisms and schizophrenia. In our population, there is no evidence that the IL1 beta gene is involved in schizophrenia.

Adult↗
PubMed1995

Characterization of the human tryptophan hydroxylase gene promoter. Transcriptional regulation by cAMP requires a new motif distinct from the cAMP-responsive element.

We isolated and sequenced 2,117 nucleotides of the promoter region of the human tryptophan hydroxylase (TPH) gene. Transient transfection in pinealocyte cultures and PC12 cells was used to investigate the human TPH (hTPH) gene promoter activity and its regulation by the cAMP signaling pathway. A region of 2,117 base pairs upstream of the transcription initiation site of the hTPH gene efficiently directed the transcription of a luciferase reporter gene but not in a cell-specific manner. The hTPH promoter activity was significantly enhanced by a cyclic AMP analog in the two cell types. Deletion analysis showed that the promoter region from -73 to +2 is sufficient to direct cAMP-dependent transcription, although it does not contain a motif exhibiting a significant identity to the cAMP-responsive element (CRE) or AP-2 binding site. Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP inducibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleotide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP responsiveness of hTPH gene expression is mediated by a cis-acting element, which shares strong identity with an inverted CCAAT box and which binds to a constitutively produced nuclear factor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Tyrosine hydroxylase regulation in neurotransmission and neuroplasticity.

The modulation of neurotransmitter synthesis is a fundamental mechanism influencing neurotransmission and neuronal plasticity during development. The regulation of the tyrosine hydroxylase (TH) has been used to elucidate specific adaptative responses in neurons. Trans-synaptic impulse activity elicits sort- and long-term changes in the activity of TH. Acute regulation involves the activation of preexisting TH molecules via phosphorylation and possibly through alternative splicing events in humans, whereas long-term regulation results from an increased synthesis of the enzyme due in part to the transcriptional stimulation of the TH gene. The long-term increase of TH activity was addressed using the drug reserpine known to modify the secretion of neurotransmitters and the tetradecanoyl phorbol acetate (TPA). Inductions of TH expression by reserpine in vivo as well as by TPA in vitro seem to be mediated by an AP-1 complex acting on a TPA responsive element (TRE) of the rat TH promoter indicating that the TRE-TH site plays a critical role in trans-synaptic induction. Our results also demonstrate a degree of adaption by sympathetic neurons to their environment by conversion from adrenergic to cholinergic phenotype.

Cells, Cultured↗