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Biomedical subjects

Y Goumon

Publications and source records attributed to Y Goumon.

At least 19 recordsLinked to original sources
PubMed2001

Presence of morphine and morphine-6-glucuronide in the marine mollusk Mytilus edulis ganglia determined by GC/MS and Q-TOF-MS. Starvation increases opiate alkaloid levels.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Mytilus edulis pedal ganglia at a level of 2.67+/-0.44 and 0.98+/-0.14 ng/ganglia, respectively by high performance liquid chromatography coupled to electrochemical detection. These opiate alkaloids were further identified by both gas-chromatography mass spectrometry and nanoflow electrospray ionization double quadrupole orthogonal acceleration Time of Flight mass spectrometry. In animals that were starved, the morphine level rose to 6.38+/-0.88 ng/ganglion and the morphine 6-glucoronide rose to a level of 23.0+/-3.2 ng/ganglion after 30 days. These studies demonstrate that opiate alkaloids are present as naturally occurring signal molecules whose levels respond to stress, i.e., starvation. Opiate alkaloids were not found in the animal's incubation media or food, demonstrating their synthesis occurred in the respective tissue. These new method of opiate alkaloid detection, conclusively proves that morphine and morphine-6-glucuronide are present in animal tissues.

Animals↗
PubMed2001

The presence of morphine in ganglionic tissues of Modiolus deminissus: a highly sensitive method of quantitation for morphine and its derivatives.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Modiolus deminissus pedal ganglia at a level of 2.41 and 0.95 ng/ganglia, respectively. These opiate alkaloids are normally found at low concentrations in invertebrate and vertebrate tissues, including neural. Given this problem, we also describe a new opiate extraction protocol as well as a high-performance liquid chromatography purification procedure that can separate and quantify morphine and its derivatives at sub-nanogram concentrations. Furthermore, both morphine and morphine-6-glucuronide were identified in this mollusk's pedal ganglia by mass spectrometry analysis.

Animals↗
PubMed2001

Presence of endogenous morphine and morphine 6 glucuronide in human heart tissue.

Human atria contain the opiate alkaloids morphine and morphine 6 glucuronide as determined by high performance liquid chromatography coupled to electrochemical detection. This method found endogenous morphine and morphine 6 glucuronide at 106.28+/-61.58 and 48.32+/-24.63 (+/- SD) ng/gm wet weight, respectively. Identification of these opiates was confirmed by nano electrospray ionization double quadrupole orthogonal acceleration time of flight mass spectrometry. Furthermore, human saphenous vein fragments did not contain morphine as determined by these methods. Fragmentation from a selected precursor ion by collision-induced dissociation of endogenous morphine 6 glucuronide (462.14 da) yields morphine (286.14 Da) obtained from the heart tissues and with the authentic material, further demonstrating the presence of endogenous morphine. Thus, vascular tissues appear to contain endogenous opiate alkaloids.

Chromatography, High Pressure Liquid↗
PubMed2000

Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.

Adrenal Medulla↗
PubMed2000

Morphine suppresses complement receptor expression, phagocytosis, and respiratory burst in neutrophils by a nitric oxide and mu(3) opiate receptor-dependent mechanism.

We investigated whether morphine and fentanyl influence surface receptor expression, phagocytic activity and superoxide anion generation of neutrophils in a whole blood flow cytometric assay. Morphine suppressed complement and Fcgamma receptor expression and neutrophil function in a concentration- and time-dependent manner. Morphine-induced changes were similar to those caused by the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine and were abolished by preincubation with the NO synthase inhibitor N-nitro-L-arginine as well as naloxone. Fentanyl had no immunosuppressive effects. These results suggest that these neutrophil functions are inhibited by morphine-stimulated NO release mediated by the mu(3) opiate receptor subtype found on immunocytes.

Fentanyl↗
PubMed2000

Lipopolysaccharide increases endogenous morphine levels in rat brain.

The present study was designed to determine whether whole body injection of lipopolysaccharide increases endogenous rat brain morphine levels. High performance liquid chromatography coupled to electrochemical detection and gas-chromatography mass spectrometry reveal that the resting brain morphine level is 7.0+/-3.2ng/g wet weight. In a time dependent manner, intraperitoneal injection of lipopolysaccharide (10 microg), leads to an increase of brain morphine level with a peak reaching after 36h post injection. After 72h following the lipopolysaccharide-injection, morphine levels increased to 66.0+/-5.4ng/g brain wet weight (P<0.001). Furthermore, 96h of fasting also increased the brain morphine level to 44.0+/-3. 6ng/g of brain wet weight. These results suggest that rat brain morphinergic processes respond to organismic challenges after a significant latent period.

Animals↗
PubMed2000

Identification of morphine in the adrenal medullary chromaffin PC-12 cell line.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.

Adrenal Gland Neoplasms↗
PubMed2000

The presence of antibacterial and opioid peptides in human plasma during coronary artery bypass surgery.

Antibacterial peptides, found in both invertebrates and vertebrates, represent a potential innate defense mechanism against microbial infections. However, it is unknown whether this process occurs in humans during surgery. We looked for evidence of release of antibacterial peptides during coronary artery bypass grafting (CABG). We used immunological techniques and antibacterial assays combined with high-performance gel-permeation chromatography, reverse-phase HPLC, N-terminal sequencing and comparison with synthetic standards to characterize the peptide B/enkelytin. We show the presence of anionic antibacterial peptide, the peptide B/enkelytin which correspond to the C-terminal part of proenkephalin A, from the plasma of patients undergoing CABG. Our studies show that peptide B/enkelytin is initially present at low levels in plasma and is then released in increased amounts just after skin incision. Antibacterial assays confirmed that the peptides specifically target gram-positive bacteria. We also demonstrate that peptide B/enkelytin is metabolized in vivo to the opioid peptides methionine-enkephalin-Arg-Phe and methionine-enkephalin, peptides that we show have granulocyte chemotactic activity. These findings suggest that in humans, surgical incision leads to the release of antibacterial peptides. Furthermore, these antibacterial peptides can be metabolized into compounds that have immune-activating properties.

Amino Acid Sequence↗
PubMed2000

Rebound from nitric oxide inhibition triggers enhanced monocyte activation and chemotaxis.

Exposure of human peripheral blood monocytes to the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) resulted in a rapid shift in cellular conformation of spontaneously activated cells from ameboid to round. The population of activated cells, approximately 7. 1 +/- 1.2%, was reduced 7-fold to 1.1 +/- 0.4% following 0.5 h exposure to SNAP. Observation of monocytes for 6 h demonstrated a gradual release from NO inhibition initiating at 2.5 h following SNAP treatment and a period of hyperactivity that was maximal at approximately 5 h following SNAP exposure. During the rebound from the NO inhibition phase, there was a significant increase in the population of activated monocytes and an increased responsiveness to chemotactic agents such as IL-1, IL-8, and fMLP relative to that of cells treated with the chemotactic agents alone. Conformational changes induced by SNAP were associated with a reduction in F-actin and loss of filopodial extension. The loss and recovery of F-actin staining paralleled changes in cell activity, suggesting that NO may alter cellular activity by modulation of cytoskeletal actin. These data taken together suggest that inhibition of monocyte activity by NO results in an excitatory phase observed subsequent to release from NO inhibition and increased sensitivity to chemotactic agents. We propose that this rebound from NO inhibition may provide increased immunosurveillance to rectify immunological problems that have been encountered during the period of inhibition.

Cell Migration Inhibition↗
PubMed2000

Ascaris suum, an intestinal parasite, produces morphine.

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 +/- 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals' nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal mu receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.

Animals↗
PubMed2000

Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17beta-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17beta-estradiol conjugated to bovine serum albumin (E(2)-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17beta-estradiol and E(2)-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17beta-estradiol-stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca(2+)]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N', N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca(2+)]i chelator, reduced [Ca(2+)]i in response to E(2)-BSA, and depleting [Ca(2+)]i stores abolished the effect of 17beta-estradiol on NO release. Confocal photomicrographs using E(2)-BSA-FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase-polymerase chain reaction, human neutrophil granulocytes expressed ERalpha but not ERbeta, suggesting that ERalpha may be the membrane receptor for 17beta-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca(2+)]i. (Blood. 2000;95:3951-3958)

Animals↗
PubMed2000

Identification of morphine in the rat adrenal gland.

Morphine was identified in rat adrenal extracts by reverse-phase HPLC, following liquid and solid extraction. All experiments were carefully performed to prevent exogenous morphine contamination. The morphine extracted from adrenal tissue (105.31 ng/g of wet adrenal gland) was identical to that of a morphine internal standard. The morphine corresponding HPLC fractions were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. The study conclusively proves morphine is endogenous to the rat adrenal gland.

Adrenal Glands↗
PubMed2000

Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A.

Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 microM range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized. In addition, from the comparison with the activity displayed by related peptides (human recombinant and rat synthetic fragments), we could determine that antibacterial and antifungal activities have different structural requirements. To assess for such activities in vivo, CGA and CGA-derived fragments were identified in secretory material released from human polymorphonuclear neutrophils upon stimulation. Vasostatin-1, which is stored in a large variety of cells (endocrine, neuroendocrine, and neurons) and which is liberated from stimulated chromaffin and immune cells upon stress, may represent a new component active in innate immunity.

Amino Acid Sequence↗
PubMed2000

Proenkephalin A-derived peptides in invertebrate innate immune processes.

Lipopolysaccharides (LPS) injection into the coelomic fluid of the leech Theromyzon tessulatum stimulates release of proenkephalin A (PEA)-derived peptides as determined by immunoprecipitation and Western blot analyses. This release occurs in the first 15 min after LPS exposure and yields a 5.3-kDa peptide fragment corresponding to the C-terminal part of the precursor. This fragment is then cleaved to free an antibacterial peptide related to mammals arginine phenylalanine extended enkelytin: the peptide B. These PEA processing peptides were characterized using a combination of techniques including reversed-phase HPLC, microsequencing and mass spectrometry. The isolated invertebrate peptide B presents a high sequence homology with the bovine's and the same activity against Gram+bacteria. Titrations revealed the simultaneous appearance of Methionine-enkephalin (ME) and peptide B in invertebrates after stimulation by LPS (in a dose-dependent manner), surgical trauma or electrical stimulations to neural tissues of the mussel. Furthermore, peptide B processing in vitro yields Methionine-enkephalin arginine phenylalanine (MERF), which exhibits via the delta receptors, immunocyte excitatory properties, i.e., movement and conformational changes, but no antibacterial activity. We surmise that this unified response to the various stimuli is a survival strategy for organism by providing immediate antibacterial activity and immunocyte stimulation, thereby reducing any immune latency period needed for an adequate immune response.

Amino Acid Sequence↗
PubMed2000

Endogenous morphine.

It is now well accepted that endogenous morphine is present in animals, both in invertebrates and vertebrates. It is a key signaling molecule that plays an important role in downregulating physiological responses, such as those in the immune system, including immune elements in the CNS. It has been demonstrated that a specific mu-opiate-receptor subtype, mu3, mediates these downregulatory effects through release of NO. This article examines morphine as an endogenous signaling molecule, in terms of its role in neural and immune regulation.

Animals↗
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↗