Biomedical subjects

Y Goumon

Publications and source records attributed to Y Goumon.

28 records · Page 2Linked to original sources

PubMed2000

Basal nitric oxide limits immune, nervous and cardiovascular excitation: human endothelia express a mu opiate receptor.

Nitric oxide (NO) is a major signaling molecule in the immune, cardiovascular and nervous systems. The synthesizing enzyme, nitric oxide synthase (NOS) occurs in three forms: endothelial (e), neuronal (n) and inducible (i) NOS. The first two are constitutively expressed. We surmise that in many tissues there is a basal level of NO and that the actions of several signaling molecules initiate increases in cNOS-derived NO to enhance momentary basal levels that exerts inhibitory cellular actions, via cellular conformational changes. It is our contention that much of the literature concerning the actions of NO really deal with i-NOS-derived NO. We make the case that cNOS is responsible for a basal or 'tonal' level of NO; that this NO keeps particular types of cells in a state of inhibition and that activation of these cells occurs through disinhibition. Furthermore, naturally occurring signaling molecules such as morphine, anandamide, interleukin-10 and 17-beta-estradiol appear to exert, in part, their beneficial physiological actions, i.e., immune and endothelial down regulation by the stimulation of cNOS. In regard to opiates, we demonstrate the presence of a human endothelial mu opiate receptor by RT-PCR and sequence determination, further substantiating the role of opiates in vascular coupling to NO release. Taken together, cNOS derived NO enhances basal NO actions, i.e., cellular activation state, and these actions are further enhanced by iNOS derived NO.

Animals↗

PubMed2000

Endogenous morphine is produced in response to cardiopulmonary bypass in neonatal pigs.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response. Endogenous morphine production has previously been demonstrated in humans after cardiac surgery with CPB. It has been hypothesized that morphine plays a role as an anti-inflammatory mediator in the systemic inflammatory response. The aim of this study was to investigate if the CPB procedure in itself elicits an endogenous morphine production in neonatal pigs. METHODS: Endogenous morphine production was measured in arterial blood in piglets exposed to sternotomy alone (sham group, n=10) or sternotomy and CPB (n=10). Blood samples were obtained immediately after the induction of anaesthesia, at the end of CPB and 4 h later. Morphine in arterial blood was detected by radioimmunoassay and confirmed by gas chromatography mass spectrometry. RESULTS: Animals undergoing CPB showed detectable endogenous morphine concentrations immediately after CPB, with increased concentrations postoperatively. There was no measurable morphine production in the sham operated pigs. CONCLUSION: The CPB procedures elicits an endogenous morphine production in neonatal pigs. This morphine response is analogous to the previously demonstrated response in patients subjected to cardiac surgery and CPB.

Animals↗

PubMed2000

Morphine inhibits NF-kappaB nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism.

BACKGROUND: The transcription factor NF-kappaB plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-kappaB-mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils. METHODS: The influence of morphine on NF-kappaB activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-kappaB was used and detected by fluoresceine-isothiocyanate-labeled anti-immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 microm, 1 mm) and incubation intervals (10-150 min) were used. RESULTS: Morphine inhibits lipopolysaccharide-induced NF-kappaB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors Nomega-nitro-l-arginine-methyl-esther and Nomega-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-kappaB nuclear binding, demonstrating that the inhibitory action is mediated by NO release. CONCLUSION: Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-kappaB nuclear binding.

Acetylcysteine↗

PubMed2000

Nociceptin, endomorphin-1 and -2 do not interact with invertebrate immune and neural mu 3 opiate receptor.

AIM: To determine if endomorphin-1, -2 and nociceptin (orphanin FQ) bind to the mu 3 opiate receptor subtype or release nitric oxide as mu 3 selective ligands do. METHODS: These opioid peptides were examined for their ability to displace [3H]dihydromorphine (DHM) binding from the invertebrate (immunocytes and pedal ganglia) mu 3 opiate receptor in membrane homogenates. The ligands were also tested for their ability to release nitric oxide from the same intact tissues utilizing an amperometric probe that measures nitric oxide in real-time. RESULTS: Endomorphin-1, -2 and nociceptin do not displace [3H]DHM binding from immunocyte or pedal ganglia membrane homogenates nor do they release nitric oxide from these tissues. CONCLUSION: Since these newly discovered opioid peptides do not interact with the mu 3 opiate receptor subtype, endogenous morphine's significance is enhanced because it appears to be the only naturally occurring opiate ligand for the receptor. Furthermore, since this study involves invertebrate tissues, this signal system had to evolve early during evolution.

Analgesics, Opioid↗

PubMed1999

A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245).

Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198-245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145-245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation. It has been proved to be efficient in measuring CgA levels in patients with neuroendocrine tumours.

Amino Acid Sequence↗

PubMed1998

Solution conformation of the synthetic bovine proenkephalin-A209-237 by 1H NMR spectroscopy.

Proenkephalin-A has been described to generate enkephalins, opoid peptides, and several derived peptides, which display various biological effects, including antinociception and immunological enhancement. Recently, we have isolated from bovine chromaffin granules a new antibacterial peptide, named enkelytin, which corresponds to the bisphosphorylated form of PEAP209-237 (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). In this paper, the three-dimensional solution structure of synthetic PEAP209-237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an alpha-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP209-237. In both models, the proline residue Pro-227 induces a 90 degrees hinge between two alpha-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall L-shaped structure for the molecule. The negative charge of PEAP209-237 and the low amphipathy of the two alpha-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin.

Amino Acid Sequence↗

PubMed1998

Characterization of antibacterial COOH-terminal proenkephalin-A-derived peptides (PEAP) in infectious fluids. Importance of enkelytin, the antibacterial PEAP209-237 secreted by stimulated chromaffin cells.

Proenkephalin-A (PEA) and its derived peptides (PEAP) have been described in neural, neuroendocrine tissues and immune cells. The processing of PEA has been extensively studied in the adrenal medulla chromaffin cell showing that maturation starts with the removal of the carboxyl-terminal PEAP209-239. In 1995, our laboratory has shown that antibacterial activity is present within the intragranular chromaffin granule matrix and in the extracellular medium following exocytosis. More recently, we have identified an intragranular peptide, named enkelytin, corresponding to the bisphosphorylated PEAP209-237, that inhibits the growth of Micrococcus luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). As a continuation of this previous study, in order to characterize the biological function of antibacterial PEAP, we have here examined whether this COOH-terminal fragment is released from stimulated chromaffin cells and whether it could be detected in wound fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were tested. We also propose a computer model of synthetic PEAP209-237 deduced from 1H NMR analysis, in order to relate the antibacterial activity of enkelytin with the three-dimensional structure. Finally, we report the high phylogenetic conservation of the COOH-terminal PEAP, which implies some important biological function and we discuss the putative importance of enkelytin in the defensive processes.

Adrenal Glands↗

PubMed1998

Antibacterial peptides are present in chromaffin cell secretory granules.

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation. 2. The agents responsible for the inhibition of Gram+ and Gram- bacteria growth are granular peptides acting in the micromolar range or below. In secretory granules, these peptides are generated from cleavage of chromogranins and proenkephalin A and are released together with catecholamines into the circulation. 3. Secretolytin and enkelytin are the best characterized; these two peptides share sequence homology and similar antibacterial activity with insect cecropins and intestinal diazepam-binding inhibitor. For some of the peptides derived from chromogranin A, posttranslational modifications were essential since antibacterial activity was expressed only when peptides were phosphorylated and/or glycosylated. 4. The significance of this activity is not yet understood. It may be reminiscent of some primitive defense mechanism or may serve as a first barrier to bacteria infection during stress, as these peptides are secreted along with catecholamines.

Amino Acid Sequence↗

PubMed1996

Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173-194 from bovine adrenal medullary chromaffin granules.

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Adrenal Medulla↗

PubMed1996

The C-terminal bisphosphorylated proenkephalin-A-(209-237)-peptide from adrenal medullary chromaffin granules possesses antibacterial activity.

The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity.

Adrenal Medulla↗