Characterization and location of post‐translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules

Gasnier, Claire; Lugardon, Karine; Ruh, Olivier; Strub, Jean‐Marc; Aunis, Dominique; Metz‐Boutigue, Marie‐Hélène

doi:10.1002/pmic.200300693

Cited by 17 publications

(21 citation statements)

References 57 publications

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“…Secretion efficiency was checked by Western blot analysis (see below) using a validated antibody raised against the conserved C-terminal part of chromogranin B (bovine CGB 614-626 [69]), which serves as a secretion marker [27].…”

Section: Methodsmentioning

confidence: 99%

“…Human hepatocyte extract (XenoTech) and human recombinant UGT2B7 (Sigma Aldrich) were used as positive controls. Chromogranin B (CGB) was detected using a rabbit polyclonal antibody raised against the conserved C-terminal part of chromogranin B (bovine CGB 614-626 [69]), and the signal was developed using HRP-conjugated anti-rabbit antisera (Sigma Aldrich, dilution 1∶400,000)…”

Section: Methodsmentioning

confidence: 99%

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Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

et al. 2008

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BackgroundMorphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system.Methodology and Principal FindingsMicroscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies.Conclusions/SignificanceThe presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

et al. 2008

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“…With in-gel IEF-LC-MS/MS, in addition to Ser 405 and Ser 149, we have found nine additional phosphorylation sites: Ser 182, Ser 183, Ser 259, Ser 263, Ser 311, Ser 335, Ser 377 (380), Ser 617, and Ser 626; none of these sites have been previously described in humans. Recently, the phosphorylation of bovine CGB at Ser 148, the equivalent of human Ser 183, as well as the phosphorylation of the equivalent bovine amino acid residues of human Ser 259, Ser 263, Ser 311, Ser 617 and Ser 626 has been reported [41]. However, in that study of bovine CGB, most of the phosphorylation site assignments were based mainly on MALDI-TOF peptide mass fingerprinting (PMF) analysis and NetPhos phosphorylation predictions [42].…”

Section: Chromogranins/secretograninsmentioning

confidence: 98%

Phosphoproteomic analysis of the human pituitary

et al. 2006

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The pituitary is the central endocrine gland that regulates the functions of various target organs in the human body. Because of the pivotal regulatory role of the pituitary, it is essential to define on a global scale the components of the pituitary protein machinery, including a comprehensive characterization of the post-translational modifications of the pituitary proteins. Of particular interest is the examination of the phosphorylation status of the pituitary in health and disease. Towards the goal of global profiling of pituitary protein phosphorylation, we report here the application of the in-gel IEF-LC-MS/MS approach to the study of the pituitary phosphoproteome. The analytical strategy combined isoelectric focusing in immobilized pH gradient strips with immobilized metal ion affinity chromatography and mass spectrometry. With this method, a total of 50 phosphorylation sites were characterized in 26 proteins. Because the investigation involved primary tissue, the findings provide a direct glimpse into the phosphoprotein machinery operating within the human pituitary tissue microenvironment.

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“…Samples purified by HPLC were loaded to polybrene‐treated glass‐fiber filters. Phenylthiohydantoin‐amino acids (Pth‐Xaa) were identified by chromatography on a C 18 column (PTH C‐18, 2.1 mm × 200 mm) (Gasnier et al, 2004). For the identification of the sequence SWISS‐Prot database was used by Blast software.…”

Section: Methodsmentioning

confidence: 99%

Modification of macroporous titanium tracheal implants with biodegradable structures: Tracking in vivo integration for determination of optimal in situ epithelialization conditions

Vrana¹,

Dupret‐Bories²,

Bach³

et al. 2012

Biotech & Bioengineering

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Previously, we showed that macroporous titanium implants, colonized in vivo together with an epithelial graft, are viable options for tracheal replacement in sheep. To decrease the number of operating steps, biomaterial-based replacements for epithelial graft and intramuscular implantation were developed in the present study. Hybrid microporous PLLA/titanium tracheal implants were designed to decrease initial stenosis and provide a surface for epithelialization. They have been implanted in New Zealand white rabbits as tracheal substitutes and compared to intramuscular implantation samples. Moreover, a basement membrane like coating of the implant surface was also designed by Layer-by-Layer (LbL) method with collagen and alginate. The results showed that the commencement of stenosis can be prevented by the microporous PLLA. For determination of the optimum time point of epithelialization after implantation, HPLC analysis of blood samples, C-reactive protein (CRP), and Chromogranin A (CGA) analyses and histology were carried out. Following 3 weeks the implant would be ready for epithelialization with respect to the amount of tissue integration. Calcein-AM labeled epithelial cell seeding showed that after 3 weeks implant surfaces were suitable for their attachment. CRP readings were steady after an initial rise in the first week. Cross-linked collagen/alginate structures show nanofibrillarity and they form uniform films over the implant surfaces without damaging the microporosity of the PLLA body. Human respiratory epithelial cells proliferated and migrated on these surfaces which provided a better alternative to PLLA film surface. In conclusion, collagen/alginate LbL coated hybrid PLLA/titanium implants are viable options for tracheal replacement, together with in situ epithelialization.

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Characterization and location of post‐translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules

Cited by 17 publications

References 57 publications

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

Phosphoproteomic analysis of the human pituitary

Modification of macroporous titanium tracheal implants with biodegradable structures: Tracking in vivo integration for determination of optimal in situ epithelialization conditions

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