Biomedical subjects

G Patey

Publications and source records attributed to G Patey.

At least 19 recordsLinked to original sources

PubMed2000

Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro.

The NT2 cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristic of a committed neuronal precursor at an early stage of development. NT2 cells can be induced by retinoic acid to differentiate in vitro into postmitotic central nervous system (CNS) neurons (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible. Because it may be possible to transplant these human neurons to compensate for neuronal loss after traumatic injuries or neurodegenerative diseases of the CNS, knowledge of their phenotype is essential. This study aimed to characterize in detail the neurotransmission phenotype of NT2-N cells by using immunocytochemical methods. Single peroxidase immunostaining demonstrated that NT2-N cells expressed the gamma-aminobutyric acidergic (GABAergic), catecholaminergic, and cholinergic phenotypes to a large extent and expressed the serotonergic phenotype to a minor extent. NT2-N cells also expressed different neuropeptides, such as neuropeptide Y, oxytocin, vasopressin, calcitonin gene-related peptide, and Met- and Leu-enkephalin. Double fluorescence immunostaining further indicated that a large number of NT2-N cells could express GABA and another neurotransmitter or neuropeptide at the same time. Finally, electron microscopy demonstrated that these NT2 neurons elaborate classical synaptic contacts. The multipotentiality of these neurons, combined with their apparent functionality, suggests that they may represent useful material for a variety of therapeutic approaches aimed at replacing dead neurons after neurodegenerative diseases or lesions of the CNS.

Cell Differentiation↗

PubMed2000

Differential expression of Bcl-2-related proteins in differentiating NT2 cells.

Although the role of Bcl-2-related proteins as regulators of the apoptotic process has been well documented, recent studies suggest that they might also be implicated in neuronal differentiation. We have studied by immunocytochemistry, Western blotting and RT-PCR the expression pattern of Bcl-xL, Bcl-2 and BAX in the in vitro model of neuronal differentiation constituted by retinoic acid (RA)-treated NTera-2/D1 (NT2/D1) cells. Whereas BAX level did not change significantly during the RA treatment, Bcl-xL level increased markedly during the first week, before returning to basal level during the second week. Bcl-2 expression, undetectable in undifferentiated cells, increased progressively from the first week. From our results, we suggest that, at least in our model, Bcl-2-related proteins might be involved in neuronal differentiation.

Apoptosis↗

PubMed1997

Immunolocalization of Bcl-xL/S in the central nervous system of neonatal and adult rats.

A polyclonal antibody raised against a peptide corresponding to the (2-19) amino-terminal sequence of the Bcl-xL/S protein was used to localize Bcl-x immunostaining in the central nervous system of rats at various postnatal ages. Whereas Bcl-x immunostaining was present in virtually all neurons of young animals (4 days postnatal), this staining became progressively restricted during the course of postnatal development. In adults, Bcl-x immunostaining was particularly strong in certain neurons present in a few hypothalamic nuclei, such as the supraoptic or the arcuate nuclei. Moderate staining was observed in some discrete brain regions, such as the olfactory bulb, the hippocampus, some catecholaminergic nuclei of the brainstem, and the cerebellum. Strong Bcl-x immunostaining was also exhibited in axon-like fibers located in the pyriform cortex, the median eminence, the dorsal medulla oblongata, and spinal cord. Bcl-x immunostaining was also present in astrocytes scattered throughout the white matter in the brain and the spinal cord, but was absent from those located in gray matter. Staining was particularly strongly expressed in reactive astrocytes densely packed along the borders of a central lesion or surrounding them, and in a large number of reactive astrocytes detected at a distance from the lesion. Our data suggest that, in addition to the possible stimulating effects on cell survival generally ascribed to Bcl-x, its maintained expression throughout adulthood or its re-expression following injury characterizes those neuronal or non-neuronal cells of the adult central nervous system that synthesize a range of molecules enabling them to adapt rapidly and successfully to a changing environment.

Animals↗

PubMed1995

Presence of the long and the short forms of Bcl-X in several human and murine tissues.

Bcl-X protein, which is related to Bcl-2, is present essentially in the nervous system and the immune system, under 2 different forms: Bcl-XL, like Bcl-2, is able to prevent apoptotic death of cultured lymphocytes; in contrast, Bcl-XS, which differs from the former one by a deletion of 63 amino acids in the region of greatest homology between Bcl-XL and Bcl-2, is unable to prevent this apoptotic death and even counteracts the protective effect of Bcl-2. It has been reported that the adult nervous system contains exclusively the RNA encoding Bcl-XL whereas the immune system would contain exclusively the RNA encoding Bcl-XS. We show here, by using reverse transcription coupled to PCR, that adult rat brain contains not only the RNA encoding Bcl-XL but also the RNA encoding Bcl-XS. Similarly we show that human lymphocytes, whether infected or not by the HIV, contain both forms of Bcl-X messenger RNA (mRNA). Furthermore we have observed that both forms of mRNA are present in adult rat liver and in cultured mouse hepatocytes. All these results have been confirmed by hybridizing the PCR products transferred to a nylon membrane with a probe specific of Bcl-XL or a non discriminative probe.

Animals↗

PubMed1992

Functional characterization of the promoter of pp63, a gene encoding a natural inhibitor of the insulin receptor tyrosine kinase.

PP63 is a liver specific phosphorylated glycoprotein encoded by a single copy gene, which has the property of inhibiting both autophosphorylation and tyrosine kinase activity of the insulin receptor. In this study, we have analyzed the structure activity relationship of the pp63 gene promoter. Five protein binding sites were found in the proximal 5' flanking region of the gene (-223 to +4). Using oligonucleotides as competitors and purified recombinant C/EBP in footprinting and gel retardation assays, we identified two typical C/EBP sites (X1 and X3) plus a heterogenous, C/EBP-NF1 like site (X5), separated by two classical NF1 binding sites (X2 and X4). C/EBP or the related proteins were predominantly involved in supporting cell-free transcription. Occupancy of the first high affinity C/EBP site conferred almost maximal promoter efficiency, in vitro. However, this pp63 promoter activity remained very low as compared to that in intact hepatocytes. In these cells, occupancy of the first C/EBP (X1) and NF1 (X2) sites was already required for achieving a weak transcriptional activity. The use of the second C/EBP site (X3) strongly enhanced transcription, up to 60-70% of the maximum, whereas occupancy of the two more distal sites (X4 and X5) was necessary to fully activate the promoter. Thus, the strength of the promoter as well as the liver specific expression of pp63 gene appear to result from the interplay of several DNA-protein complexes involving mainly C/EBP and/or related proteins as well as the ubiquitous NF1 factor(s), rather than from the interaction of a more liver specific trans-acting factor with the promoter.

Animals↗

PubMed1991

Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase.

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.

Animals↗

PubMed1989

Molecular characterization of the human beta 3-adrenergic receptor.

Since the classification of beta-adrenergic receptors (beta-ARs) into beta 1 and beta 2 subtypes, additional beta-ARs have been implicated in the control of various metabolic processes by catecholamines. A human gene has been isolated that encodes a third beta-AR, here referred to as the "beta 3-adrenergic receptor." Exposure of eukaryotic cells transfected with this gene to adrenaline or noradrenaline promotes the accumulation of adenosine 3',5'-monophosphate; only 2 of 11 classical beta-AR blockers efficiently inhibited this effect, whereas two others behaved as beta 3-AR agonists. The potency order of beta-AR agonists for the beta 3-AR correlates with their rank order for stimulating various metabolic processes in tissues where atypical adrenergic sites are thought to exist. In particular, novel beta-AR agonists having high thermogenic, antiobesity, and antidiabetic activities in animal models are among the most potent stimulators of the beta 3-AR.

Adrenergic beta-Agonists↗

PubMed1987

Release of [Met]enkephalin in the central nucleus of the amygdala is increased by application of potassium in the substantia nigra.

Release of [Met]enkephalin immunoreactivity (Met-IR) in the central nucleus of the amygdala (ACE) was investigated in vivo in anesthetized rats implanted with push-pull cannulae. A stable spontaneous release of this peptide (1.3 fmol/15 min fraction) could be measured in the superfusates using a highly sensitive radioimmunoassay. The addition to the superfusion medium of cocktail of peptidase inhibitors increased three times the spontaneous release of the peptide. Superfusion with 30 mM potassium increased ten times the release of the peptide. Chemical stimulation of the substantia nigra with K+ enhanced four times the Met-IR release in the ipsilateral ACE. The dopaminergic component of the nigro-amygdaloid pathway appeared not to be directly implicated in this effect, since: d(+)amphetamine application in the ACE, which enhanced the local release of DA, remained without effect on Met-IR release and haloperidol-induced blockade of dopaminergic receptors in the ACE similarly did not affect Met-IR release.

Amygdala↗

PubMed1986

[Discovery, anatomical mapping and biosynthesis of various families of endogenous opioid peptides].

The endogenous opioid peptides all contain the enkephalin sequence Tyr-Gly-Gly-Phe (-Met/-Leu at their amino-terminus. Three distinct families of these peptides (beta-endorphins, enkephalins and dynorphins) are present in different neuronal pathways within the central nervous system. Molecular genetics have shown that these three families of opioid peptides are derived from three distinct precursors. Pro-opiomelanocortin gives rise to the endorphins, as well as adrenocorticotropic hormone (ACTH) and the melanotropic hormones (MSH's). Met-enkephalin, Leu-enkephalin and the related heptapeptide Met-enkephalin-Arg6-Phe7 and octapeptide Met-enkephalin-Arg6-Gly7-Leu8 are derived from proenkephalin. The third family is derived from prodynorphin and includes dynorphin A, dynorphin B (also known as rimorphin) and alpha- and beta-neo-endorphin. The structures of the genes coding for these precursors are similar, suggesting the possibility of one common ancestral gene. At the present time the main question concerns the physiological significance of such a great diversity of endogenous opioid peptides.

Brain Chemistry↗

PubMed1985

Characterization of a new 23 kDalton enkephalin-containing protein in the bovine adrenal medulla.

Immunoblots combined with specific radioimmunoassays (RIAs) have been used to visualize simultaneously all the enkephalin-containing peptides (ECPs) present in a crude extract of bovine adrenal medulla. They have allowed the characterization of a new high molecular weight ECP which has a molecular weight of 23.3 kDalton, contains the amino-terminal part of proenkephalin and ends with the sequence of Leu-enkephalin at its carboxy-terminus.

Adrenal Medulla↗

PubMed1985

Release of proenkephalin-derived opioid peptides from rat striatum in vitro and their rapid degradation.

In a previous paper we demonstrated that the heptapeptide [Met]enkephalyl-Arg6-Phe7 was released from rat striatal slices by high K+ concentration and rapidly degraded by peptidases, even in the presence of the neutral endopeptidase 24.11 ("enkephalinase")-inhibitor, thiorphan (0.1 microM), the angiotensin-converting enzyme inhibitor, captopril (1 microM), and the aminopeptidase inhibitor, bestatin (20 microM). In this study the pattern of degradation of exogenous [3H]heptapeptide by rat striatal slices has been studied. The angiotensin-converting enzyme and aminopeptidase(s) were partly responsible for this degradation. In addition an enzymatic activity that cleaved the Phe4-Met5 bond was involved in the degradation of the heptapeptide by striatal slices. This activity was inhibited by the dipeptide Leu-Arg (1 mM) and the tripeptide Leu-Arg-Leu (1 mM). The simultaneous presence of thiorphan (0.1 microM), captopril (1 microM), bestatin (20 microM) and Leu-Arg (1 mM) almost completely inhibited the degradation of [3H]heptapeptide by striatal slices. In the presence of these peptidase inhibitors a concomitant release of [Met]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8 was evoked by KCl or veratridine. The K+-evoked release was by a Ca2+-dependent mechanism and the release evoked by veratridine was blocked by tetrodotoxin. In both cases the ratio of [Met]enkephalin to heptapeptide amounts released was close to that found in their common precursor, proenkephalin. Thus the enkephalinergic neuron appears to be capable of synthesizing, from a unique precursor, four different putative opioid neurotransmitters, namely [Met]enkephalin, [Leu]enkephalin, the heptapeptide [Met]enkephalyl-Arg6-Phe7 and the octapeptide [Met]enkephalyl-Arg6-Gly7-Leu8, to store these peptides and to release them upon depolarization.

Animals↗

PubMed1984

Processing of proenkephalin is tissue-specific.

Most neuropeptides are synthesized as large precursor proteins. These precursors undergo a maturation process involving several proteolytic events that generate the biologically active peptides. The enzymatic mechanisms underlying this processing are still largely unknown. The processing of the precursor protein proenkephalin was studied in two different bovine tissues, the hypothalamus and adrenal medulla. The high molecular weight enkephalin-containing peptides that accumulate in these two tissues were found to be different, indicating the existence of two processing pathways for this neuropeptide precursor.

Adrenal Medulla↗

PubMed1984

Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting.

Immunoblotting combined with radioimmunoassays (RIAs) directed specifically towards certain sequences of the proenkephalin molecule has been used to characterize the enkephalin-containing peptides (ECPs) present in the bovine adrenal medulla. Immunoblotting allowed the simultaneous visualization of all ECPs present in a crude extract of this gland. Combining this technique with RIAs we have been able to characterize a new high molecular mass ECP, a 23.3-kDa protein which contains the amino-terminal part of proenkephalin and ends with the sequence of Leu-enkephalin at its carboxy-terminus.

Adrenal Medulla↗

PubMed1984

Quantitation and localization of Met-enkephalin-Arg-Gly-Leu in rat brain using highly sensitive antibodies.

Met-enkephalin-Arg-Gly-Leu is an endogenous opioid peptide recently identified in bovine adrenal medulla. In the present study, we describe the production of highly sensitive and specific antibodies against this octapeptide. The sensibility of the radioimmunoassay procedure allows us to quantify at the femtomole level, the Met-enkephalin-Arg-Gly-Leu in individual parts of the brain without prior purification or concentration. The antibodies are highly specific for the C terminal part of the molecule, and did not cross-react with the other opioid peptides. Immunochemical techniques were used also to determine the histological location of the immunoreactive substances in individual structures of the brain. In the present paper, the comparative regional distribution of Met-enkephalin-Arg-Gly-Leu and of Met-enkephalin in rat brain are described. Our results are in good agreement with the biosynthetic relationship between Met-enkephalin and Met-enkephalin-Arg-Gly-Leu.

Animals↗

PubMed1983

The heptapeptide Met-enkephalin-Arg6, Phe7 is released from rat striatum in vitro by high potassium.

We present evidence that the heptapeptide Met-enkephalin-Arg6, Phe7 is released from rat striatal slices following depolarization by a high concentration of KCl. The heptapeptide-immunoreactive material released in the incubation medium, which is shown by HPLC and radioimmunoassay of serial dilutions to represent authentic heptapeptide, is detectable in the incubation media only in the presence of a cocktail of peptidase inhibitors containing thiorphan (0.1 microM), captopril (1 microM) and bestatin (20 microM).

Animals↗

PubMed1983

Release of the heptapeptide Met-enkephalin-Arg6-Phe7 and of the octapeptide Met-enkephalin-Arg6-Gly7-Leu8 from rat striatum in vitro and their rapid inactivation.

The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) and the octapeptide Met-enkephalin-Arg6-Gly7-Leu8 (MERGL) are potent opioid peptides present in the sequence of proenkephalin, the common precursor of Met- and Leu-enkephalin (ME and LE). We demonstrate that MERF and MERGL are released concomitantly with ME and LE from rat striatal slices following a depolarisation by K+. This release is a Ca2+-dependent process. While the ratios of ME to LE, MERF and MERGL found in the tissue (ME/LE = 2.6; ME/MERF = 3.1; ME/MERGL = 4.5) are in good agreement with the ratios found in the proenkephalin molecule (ME:LE:MERF:MERGL = 4:1:1:1), the amounts of MERF and MERGL recovered from the medium are low compared to those of ME and LE, suggesting a rapid degradation of released MERF and MERGL. In fact, when incubated with striatal slices, (3H-Tyr)-MERF is rapidly degraded by four classes of peptidases: the "enkephalinase", the angiotensin-converting enzyme (ACE), aminopeptidase(s) and an endopeptidase releasing the tetrapeptide Tyr-Gly-Gly-Phe (YGGF). Whereas the activities of the three former peptidases are reduced or abolished in the presence of thiorphan (0.1 microM), captopril (1 microM) and bestatin (20 microM), the amount of YGGF formed by the endopeptidase is not reduced in these conditions but actually increased.

Animals↗

PubMed1983

The enkephalinergic neuron: implications of a polyenkephalin precursor.

The study of the biochemical and physiological functions of the enkephalinergic cell has greatly extended our understanding of peptidergic cells in general. In the adrenal gland, the major part of the proenkephalin-derived peptides is present as intermediates in the processing of the precursor. These peptides are contained within the adrenergic chromaffin granules, from which they are released in response to stimulation of the cell. The nature of the products released depends on the nature of the stimulus, but it appears that mature granules containing completely processed peptides are preferentially released under physiological conditions. In the brain, the presence and release of the heptapeptide that comprises the carboxyl terminus of adrenal proenkephalin suggest that similar mechanisms are operating centrally. The identity of brain and adrenal proenkephalin is further supported by the purification from brain of a large fragment of the proenkephalin molecule, synenkephalin , and the occurrence in brain of this and the other proenkephalin-derived peptides in a molar ratio close to that found in the sequence of the adrenal precursor. The processing of proenkephalin in brain appears to follow the classical models first proposed for peptide hormones (Steiner et al. 1980), which may thus be generalized to include peptide neurotransmitters/neuroregulators. In addition, the results presented in this paper indicate that enkephalins may be cotransmitters in at least two diverse systems. Enkephalins and catecholamines are colocalized in the adrenergic granules of the adrenal gland. In the brain, enkephalins and oxytocin are colocalized in the magnocellular neurons of the hypothalamo-neurohypophyseal oxytocinergic pathway. In both of these systems, the enkephalins are present in a molar concentration that is less than 1% of the concentration of the principal messenger. Such colocalization , coupled with the numerous active peptides that may arise from proenkephalin, suggests many elegant but complex schemes of neurotransmitter interactions. For example, release of enkephalins in the neurohypophysis may regulate oxytocin release through an action on autoreceptors of the oxytocinergic terminal. In the adrenal the coreleased enkephalins may act by regulating presynaptically the cholinergic output of the splanchnic nerve. However, further studies are needed to define clearly the physiological roles of such cotransmission . From the abundance of proenkephalin-derived peptides in the basal ganglia, it appears that enkephalins may represent the principal transmitter in some central neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Medulla↗

PubMed1983

Regional distribution of methionine-enkephalin-Arg6-Phe7 in the rat brain: comparative study with the distribution of other opioid peptides.

The distribution of the opioid peptide methionine-enkephalin-arginine6-phenylalanine7 (M-Enk-Arg6-Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M-Enk-Arg6-Phe7 has been further characterized by high performance liquid chromatography. The levels of M-Enk-Arg6-Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionine-enkephalin (M-Enk), leucine-enkephalin (L-Enk), dynorphin 1-13, and alpha-neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M-Enk-Arg6-Phe7 immunoreactive material (ir), M-Enk ir, and L-Enk ir. The distribution of dynorphin 1-13 ir and alpha-neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c-DNA coding for the prohormones, in which it has been hypothesized that M-Enk-Arg6-Phe7 and M-Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin-related peptides and alpha-neoendorphin arise from a separate precursor, prodynorphin.

Amino Acid Sequence↗