Search PubMed⌕ Search

Biomedical subjects

P Vernier

Publications and source records attributed to P Vernier.

At least 37 records · Page 2Linked to original sources
PubMed1998

Tyrosine hydroxylase in the european eel (Anguilla anguilla): cDNA cloning, brain distribution, and phylogenetic analysis.

We report the isolation of a full-length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3' untranslated region and by a diversity restricted to the 3' end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU-rich 3' noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N-terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH-expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.

Amino Acid Sequence↗
PubMed1998

Retinoic acid regulates the developmental expression of dopamine D2 receptor in rat striatal primary cultures.

The time course of D2 receptor expression assessed by the levels of the corresponding binding sites and mRNA was studied in rat striatum during ontogenesis and in primary cultures of cells taken at embryonic day (E) 17 and postnatal day (P) 4. In the two experimental situations, the amount of D2 receptor mRNA and number of binding sites increased regularly from E16 to P15, indicating that expression of D2 receptors in striatal neurons occurs independently from a dopaminergic input. Incubation of striatal primary cultures with 10(-5) M retinoic acid significantly increased the level of D2 receptor mRNA, whereas thyroid hormone, vitamin D3, and steroid hormones (estradiol, testosterone, and corticosterone) had no effect. The transcriptional activity of the rat D2 receptor gene promoter region, which bears a retinoic acid-responsive element, was increased by retinoic acid in transfected C6 glioma cells but not in transfected MMQ prolactin cells. Thyroid hormone and vitamin D3 were not effective in either cell line. Finally, mutations of the putative retinoic acid-responsive element inhibited the transcriptional effect of retinoic acid. These results suggest that retinoic acid is a key factor in regulation of the embryonic onset of the dopaminergic D2 receptor.

Animals↗
PubMed1998

Functional differentiation of multiple dopamine D1-like receptors by NNC 01-0012.

Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH-23390 binding with an estimated affinity (approximately 0.6 nM) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by approximately 33% relative to the full agonist dopamine (10 microM), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N-methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.

Animals↗
PubMed1998

Dopamine D1B receptor chimeras reveal modulation of partial agonist activity by carboxyl-terminal tail sequences.

NNC 01-0012, a second-generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01-0012 acts as a full or poor antagonist at D1C and D1A receptor-mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01-0012, a series of receptor chimeras were constructed in which the divergent carboxyl-terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl-terminal-tail at position Tyr345 with carboxyl-terminal-tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01-0012 without affecting dopamine-stimulated cyclic AMP accumulation. At vertebrate D1B/D1CcT-tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01-0012 (10 microM) was markedly enhanced (approximately 60% relative to 10 microM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF-38393 and SCH-23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl-terminal tails with sequences encoded by the D1B receptor carboxyl-terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well-characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence-specific motifs within the carboxyl-terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1-like receptors.

Animals↗
PubMed1998

Emergence of a brain-expressed variant melanin-concentrating hormone gene during higher primate evolution: a gene "in search of a function".

Two related but distinct melanin-concentrating hormone (MCH) gene systems, i.e., the authentic and variant genes, have been characterized in the human, while only a single MCH gene has been found in the rat. We previously established that the variant gene corresponds to exon-I-deleted copies of the authentic gene mapped on chromosomes 5 and 12, respectively. In this report, we examined the expression of the authentic and variant MCH genes in the human brain. Mature mRNAs of the authentic MCH gene appeared to be predominantly expressed in the hypothalamus, whereas putative unprocessed transcripts of the variant MCH gene were found in other brain areas but not in the hypothalamus. Several products of the variant MCH gene were identified by RACE-PCR in the fetal human brain. One of these transcripts encoded a putative protein of 72 amino acids, while another transcript may potentially generate a protein of 35 amino acids. Thereafter, we explored the question of MCH gene transposition during Primate evolution. Southern blotting, PCR analyses using several genomic DNAs of Primates, and in situ hybridization on metaphase chromosomes led us to define at least three types of genetic events associated with the emergence of the variant MCH gene: (1) translocation of an exon II-exon III copy of the authentic MCH gene onto the equivalent of the human chromosome 5p arm of Anthropoidea ancestors (between 25 and 55 MYA); (2) exon II truncation and mutations before divergence of the Hylobatidae (about 15 MYA); and (3) duplication of the variant gene on the equivalent of the human chromosome 5p and 5q arms in the Hominidae, i.e., 5-10 MYA. Taken together, these results support the hypothesis that transposition/gene rearrangement processes could underlie the evolutionary emergence of new MCH-related genes expressed differentially in the brains of higher Primates, illustrating the concept of genes "in search of function" instead of true "pseudogenes."

Animals↗
PubMed1998

Alternative splicing of the D2 dopamine receptor messenger ribonucleic acid is modulated by activated sex steroid receptors in the MMQ prolactin cell line.

The two isoforms of the D2 dopamine receptor are generated by alternative splicing of the exon 6 of the premessenger RNA (pre-mRNA), changing the length of the third cytoplasmic loop involved in the coupling to G proteins. In the MMQ PRL cell line, sex steroid hormones modulated the proportion of the two D2 receptor isoforms. Under controlled culture conditions, 17beta-estradiol (E2) strongly favored the production of the long isoform of D2 mRNA over the short one, whereas both isoforms were equally abundant when culture medium was hormone depleted. In the presence of progesterone (P), E2 action was inhibited, and equal amounts of each D2 receptor isoform were produced in the cells. Hormone treatments never modified either the total amount of D2 receptor mRNA and D2 receptor binding sites or D2 receptor-mediated inhibition of adenylyl cyclase. Specific antagonists demonstrated that the activity of each hormone depended on their nuclear receptors. Inhibitors of gene transcription or translation also showed that their activity required protein synthesis. The expression of the short D2 receptor isoform was never prominent, even at the single cell level. Analysis of the intron sequence flanking alternative exon 6 showed that only the upstream intron presented two sequence tracts known to be targets for splicing factors. Taken together, these results provide converging evidence for a physiologically relevant mechanism by which sex steroid receptors could regulate the expression of a splicing factor favoring the production of the long dopamine D2 receptor isoform.

Alternative Splicing↗
PubMed1998

[Evolution of monoamine receptors and the origin of motivational and emotional systems in vertebrates].

The evolving vertebrate nervous system was accompanied by major gene duplication events generating novel organs and a sympathetic system. Vertebrate neural pathways synthesizing catecholamine neurotransmitters (dopamine and noradrenaline), were subsequently recruited to process increased information demands by mediating psychomotor functions such as selective attention/predictive reward and emotional drive via the activation of multiple G-protein linked catecholamine receptor subtypes. Here we show that the evolution of these receptor-mediated events were similarly driven by forces of gene duplication, at the cephalochordate/vertebrate transition. In the cephalochordate Amphioxus, a sister group to vertebrates, a single catecholamine receptor gene was found, which based on molecular phylogeny and functional analysis formed a monophyletic group with both vertebrate dopamine D1 and beta adrenergic receptor classes. In addition, the presence of dopamine but not of noradrenaline was assayed in Amphioxus. In contrast, two distinct genes homologous to jawed vertebrate dopamine D1 and beta adrenergic receptor genes were extant in representatives of the earliest craniates, lamprey and hagfish, paralleling high dopamine and noradrenaline content throughout the brain. These data suggest that a D1/beta receptor gene duplication was required to elaborate novel catecholamine psychomotor adaptive responses and that a noradrenergic system specifically emerged at the origin of vertebrate evolution.

Animals↗
PubMed1997

Molecular identification of a dopamine D1b receptor in bovine retinal pigment epithelium.

The rhythmic daytime inhibition of phagocytosis of shed photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) is related to increased cAMP in RPE cells. Dopamine (DA), the light-adaptive signal of the retinal oscillator can activate adenylyl cyclase through its D1-like receptors. It reduces OS phagocytosis by cultured bovine RPE, but a DA receptor was not demonstrated. Using primers selected from alignment of D1-like receptor genes already cloned, we have amplified by PCR two sequences in bovine genomic DNA. Phylogenetic analysis demonstrated that they correspond to D1a and D1b receptors. These receptors were then searched for using the reverse transcription-polymerase chain reaction (RT-PCR) in cultured bovine RPE. Only the D1b receptor subtype was demonstrated. It could mediate the DA-induced inhibition of phagocytosis.

Animals↗
PubMed1997

Early emergence of three dopamine D1 receptor subtypes in vertebrates. Molecular phylogenetic, pharmacological, and functional criteria defining D1A, D1B, and D1C receptors in European eel Anguilla anguilla.

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.

Adenylyl Cyclases↗
PubMed1997

Modulation of NCAM polysialylation is associated with morphofunctional modifications in the hypothalamo-neurohypophysial system during lactation.

Post-transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell-cell interactions. Polysialic acid-NCAM (PSA-NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo-neurohypophysial system during lactation in rats. Since cell-cell interactions and synaptic contacts in the hypothalamo-neurohypophysial system are greatly increased during lactation, we examined whether PSA-NCAM expression is modified during this period. Immunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA-NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo-neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule-associated protein-1B (MAP1B-P), whose distribution pattern largely overlaps with that of PSA-NCAM in the adult brain. Expression of MAP1B-P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA-NCAM and MAP1B-P may reflect the permanent structural plasticity characterizing the hypothalamo-neurohypophysial system in the adult.

Animals↗
PubMed1997

The c-Jun-induced transformation process involves complex regulation of tenascin-C expression.

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. We identified c-Jun-upregulated cDNA clones homologous to the tenascin-C gene by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induced tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis was repressed after stable transformation by c-Jun compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. Furthermore, as demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. We previously described two other extracellular matrix proteins, SPARC and thrombospondin-1, as c-Jun targets. Thus, our results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation.

Animals↗
PubMed1996

The major astrocytic phosphoprotein PEA-15 is encoded by two mRNAs conserved on their full length in mouse and human.

Specific phosphoproteins are targets of numerous extracellular signals received by astrocytes. One such target, which we previously described, is PEA-15, a protein kinase C substrate associated with microtubules. Two cDNAs differing in the length of their 3'-untranslated region (3'UTR) were cloned from a mouse astrocytic library. Accordingly, Northern blots revealed two transcripts (1.7 and 2.5 kilobase pairs) abundant brain regions but also found in peripheral tissues. PEA-15-deduced protein sequence (130 amino acids) shared no similarity with known proteins but is 96% identical to its human counterpart. In addition, several regions of the 3'UTR share more than 90% identity between mouse and human. Different potential regulatory sequences are found in the 3'UTR, which also completely includes the proto-oncogene MAT1. The high level of conservation of both the coding and the untranslated regions and the differential tissular distribution of the two transcripts of this major brain phosphoprotein suggest that not only the protein but also the 3'UTR of PEA-15 mRNA play a role in astrocytic functions.

Amino Acid Sequence↗
PubMed1996

Radioimager quantification of oligonucleotide hybridization with DNA immobilized on transfer membrane: application to the identification of related sequences.

The radioimager scintillating optical fiber imager was used to quantify the hybridization parameters of a 35-mer oligonucleotide probe with target DNAs immobilized on transfer membranes. The amount of the immobilized target DNA remaining accessible to hybridization (Rt) was shown to be about 4% of the spotted DNA. The time course of the hybridization of a target DNA reacting with an excess of full-match probe exhibited a first-order kinetics, in which rate constant k was the highest for the hybridization temperature close to the calculated Tm. The effect of temperature on the hybridization kinetics of the probe sharing 37 to 100% identity with the immobilized target DNA was assessed: A significant fall of both the rate constant k and Rt values at the plateau was observed when the identity shared by the target DNA and the probe decreased from 100 to 71%. The highest k and Rt values were also obtained for temperatures closest to the calculated Tm. A good estimate of the degree of sequence identity may be calculated from the corresponding hybridization signals. Washing procedure did not improve the discrimination between related sequences, except for closely similar sequences. Practical conclusions for the detection of sequences belonging to gene families are presented.

Base Sequence↗
PubMed1996

Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis.

In order to determine whether G protein-coupled receptors play a role in early embryogenesis, we looked for cDNA fragments amplified between primers located in consensus sequences of transmembrane segments. Using one such amplified fragment as a probe, we cloned a novel member of the G protein-coupled receptor superfamily in Xenopus. Alignment of the deduced protein sequence with that of other receptors discloses some homology with angiotensin receptors. A single transcript of 2.5 kb is detected at the late blastula stage and its expression increases during gastrulation. In situ hybridization reveals transcripts initially in the ventrolateral involuting marginal zone and later in the lateral plate mesoderm. At larval stages, the transcript is expressed in procardiac tube and forming blood vessels, where it is localized in the inner endothelial layer. Thus, this gene traces an endothelial lineage and represents a very early and unique marker in Xenopus of the specification of cardiac and vascular endothelia. We propose the name of X-msr for mesenchyme-associated serpentine receptor.

Amino Acid Sequence↗
PubMed1995

Double in situ hybridization reveals overlapping neuronal populations expressing the low molecular weight GTPases Rab3a and Rab3b in Rat brain.

The ras-related Rab3 gene subfamily codes for small GTP-binding proteins which control a late step of exocytosis during which vesicles become docked to the plasma membrane. Rab3a and Rab3b are the most abundant Rab3 isoforms expressed in the CNS of mammals. We have shown previously that the Rab3a protein was selectively distributed and expressed in various regions of the rat brain. Here we have determined the pattern of expression of Rab3b mRNA in the brain and compared it with that of Rab3a mRNA. In addition, we examined the co-expression of these two Rab within individual neurons. In general the Rab3b transcript was detected in many regions which also express Rab3a mRNA but at a lower level than Rab3a, except in the olfactory bulb and in the pituitary where the Rab3b hybridization signal was similar and higher respectively. Double in situ hybridization revealed that Rab3a and Rab3b mRNAs were co-localized in most neurons, in all brain areas examined. However, in each of these areas, subsets of neurons appeared to preferentially express either Rab3b or Rab3a, or some neurons did not express either Rab3 homologue at detectable levels. These data support the view of a functional specialization of Rab3a and Rab3b in the control of exocytosis in neuronal and neuroendocrine cells.

Animals↗