Biomedical subjects

D Aunis

Publications and source records attributed to D Aunis.

At least 73 records · Page 4Linked to original sources

PubMed1997

Trimeric G proteins control exocytosis in chromaffin cells. Go regulates the peripheral actin network and catecholamine secretion by a mechanism involving the small GTP-binding protein Rho.

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles and its activation by mastoparan inhibits the ATP-dependent priming of exocytosis. The effectors by which Go controls exocytosis are currently unknown. The subplasmalemmal actin network is one candidate, since it modulates secretion by controlling the movement of secretory granules to the plasma membrane. In streptolysin-O-permeabilized chromaffin cells, activation of exocytosis produces disassembly of cortical actin filaments. Mastoparan blocks the calcium-evoked disruption of cortical actin, and this effect is specifically inhibited by antibodies against Galphao and by a synthetic peptide corresponding to the COOH-terminal domain of Galphao. Disruption of actin filaments with cytochalasin E and Clostridium perfringens iota toxin partially reverses the mastoparan-induced inhibition of secretion. Furthermore, the effects of mastoparan on cortical actin and exocytosis are greatly reduced in cells treated with Clostridium botulinum C3 exoenzyme, which specifically inactivates the small G protein Rho. We propose that the control exerted by the granule-associated Go on exocytosis may be related to effects on the cortical actin network through a sequence of events which eventually involves the participation of Rho.

ADP Ribose Transferases↗

PubMed1997

Phosphorylation and O-glycosylation sites of bovine chromogranin A from adrenal medullary chromaffin granules and their relationship with biological activities.

Bovine adrenal medullary chromogranin A, the major soluble component of chromaffin granules, is a phosphorylated glycoprotein. In the present work, phosphorylation and glycosylation sites were determined using mild proteolysis, peptide separation, microsequencing, and mass analysis by electrospray and matrix-assisted laser desorption ionization time-of-flight techniques. Seven post-translational modification sites were detected. Two O-linked glycosylation sites, each consisting of the trisaccharide NeuAcalpha2-3Galbeta1-3GalNAcalpha1, were located in the middle part of the protein, on Ser186 and on Thr231. The former residue is present in the antibacterial peptide named chromacin. Four phosphorylation sites were located on serine residues at positions Ser81 in the N-terminal region of the protein and Ser307, Ser372, and Ser376 in the C-terminal end. One additional phosphorylation site was found on the tyrosine residue at position Tyr173, the N-terminal amino acid of chromacin. With the exception of the phosphorylation on Tyr173, all of the other post-translational modifications are located on highly conserved chromogranin A regions, implying some biological importance.

Adrenal Medulla↗

PubMed1997

Characterization of nuclear proteins that bind to the regulatory TGATTGGC motif in the human immunodeficiency virus type 1 long terminal repeat.

We have recently elucidated the nature and function of transcription factors present in Jurkat, glial and neuronal cells that interact with modulatory region B, the nuclear receptor responsive element, in the long terminal repeat of human immunodeficiency virus type 1 (HIV-1). Considering the key role that the combination of host cell proteins plays in HIV-1 gene transcription, it appears essential to characterize proteins interacting with the adjacent region A. In vitro experiments revealed that the 5'-TGATTGGC-3'motif of region A is the target for at least three distinct proteins, one belonging to the nuclear factor I family, while two others are related to the cAMP response element binding (CREB) protein family. One of these proteins, present in DNA-protein complex C2, is formed by distinct polypeptides of relative molecular mass 43 000 and 50 000. We have purified the 43 kDa protein, which is distinct from CREB-43, and have shown that renatured p43 is able to specifically interact with site A. Transient expression experiments with vectors containing wild-type or mutant motif A revealed that basal HIV-1 gene transcription in Jurkat cells is regulated by antagonistic effects of the site A binding proteins.

Base Sequence↗

PubMed1997

Regulated exocytosis in chromaffin cells. A potential role for a secretory granule-associated ARF6 protein.

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

ADP-Ribosylation Factors↗

PubMed1997

Immediate early gene induction by natriuretic peptides in PC12 phaeochromocytoma and C6 glioma cells.

The effect of the natriuretic peptides ANP, BNP and CNP on cGMP formation and immediate early gene expression was investigated in PC12 phaeochromocytoma and C6 glioma cell lines. The three natriuretic peptides were shown to rapidly induce c-fos, TIS8/egr-1 and junB mRNA expression in both cell lines, via stimulation of the cGMP pathway. CNP stimulated cGMP formation and gene induction more potently than the other peptides in C6 cells, and this was statistically significant. In contrast, the three peptides produced similar gene induction in PC12 cells, despite the higher cGMP accumulation evoked by ANP or BNP. CNP was also found to increase DNA binding activity of the transcription factor AP1 in both cell types, demonstrating that natriuretic peptides potentially regulate key cellular gene expression.

Animals↗

PubMed1997

Exocytosis in single chromaffin cells: regulation by a secretory granule-associated Go protein.

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolic protein GAP-43 stimulates G alpha o in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin, cells by stimulating a granule-associated Go protein.

Antibodies↗

PubMed1997

5-Hydroxytryptamine induces TIS8/egr-1 and c-fos expression in PC12 cells. Involvement of tyrosine protein phosphorylation.

The TIS8/egr-1 gene is a member of the class of immediate early genes. Originally discovered as a mitogen-induced gene, it can also be induced by synaptic activity. We report here the induction of the TIS8/egr-1 gene by the neurotransmitter 5-hydroxytryptamine (5-HT) in cultured rat phaeochromocytoma PC12 cells. Induction was maximal 40 min after addition of 5-HT to the cells, and declined very rapidly to reach the basal level after 90 min. The electrophoretic mobility-shift assay showed that induction of the TIS8/egr-1 gene by 5-HT was accompained by increased Egr-1 protein binding to its DNA consensus sequence. We found an overall correlation of 5-HT-induced egr-1 expression with that of c-fos expression. The kinetics of the ability of both gene products to bind to their respective DNA consensus sequence was also similar. The 5-HT-induced activation in Egr-1 binding was inhibited by ketanserin and mesulergine, indicating that 5-HT exerted its action via a 5-HT2 receptor subtype. The tyrosine protein kinase inhibitor genistein abolished induction of the TIS8/egr-1 gene, suggesting that tyrosine kinase activity is required for the induction of early genes by 5-HT. Genistein also inhibited 5-HT-induced Egr-1 binding activity. The increase in phosphotyrosine content of focal adhesion kinase we noticed upon addition of 5-HT to cells suggests that this cytoplasmic tyrosine protein kinase mediates the effect of 5-HT in eliciting early gene induction.

Animals↗

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

Chicken ovalbumin upstream promoter transcription factor, a transcriptional activator of HIV-1 gene expression in human brain cells.

Viral infection of the central nervous system by the human immunodeficiency virus type 1 leads to a wide range of neuropathological disorders. However, the molecular mechanisms governing transcription of the human immunodeficiency virus type 1 genome in brain remain unclear. We have recently established that in brain cells, proteins belonging to the steroid/thyroid/retinoic acid receptor family bind to the -352 to -320 region of the long terminal repeat (LTR). Here, by supershift experiments, we have identified chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of this nuclear receptor family, as one of the major proteins interacting with this LTR site. Cotransfection studies revealed that COUP-TF is able to dramatically activate LTR-directed gene transcription in human oligodendroglioma but not in astrocytoma cells. This activation occurs through two mechanisms, depending on the LTR sequence. Moreover, in neuronal cells COUP-TF and dopamine, a catecholamine neurotransmitter, enhance LTR-directed transcription by acting on the proximal LTR region. These results reveal the importance of COUP-TF and the dopamine signaling pathway as activators of human immunodeficiency virus type 1 gene expression in brain.

Brain↗

PubMed1996

Regulation of human immunodeficiency virus type 1 gene transcription by nuclear receptors in human brain cells.

Infection of cells of the central nervous system by the human immunodeficiency virus type-1 (HIV-1) leads to HIV-1-associated neuropathology. Recent studies have demonstrated the importance of long terminal repeat (LTR) binding sites in determining the pathogenicity of HIV. Here we have investigated the presence and the functional role of transcription factors that have the potential to interact, directly or indirectly, with the nuclear receptor-responsive element in the LTR of HIV-1, in different human cell lines of the brain. Cotransfection experiments showed that in oligodendroglioma TC-620 cells, the retinoic acid receptor and the retinoid X receptor activate LTR-driven transcription in the absence of ligand. Addition of all-trans- or 9-cis-retinoic acid reverses this effect. In contrast, in astrocytoma, neuronal, and microglial cells, no significant effect of the retinoid acid pathway was detected. This retinoid response is mediated by distinct molecular interactions in the lymphotropic LAI and the neurotropic JR-CSF HIV-1 strains. Moreover, retinoid receptors were found to antagonize the chicken ovalbumin upstream promoter transcription factor- as well as the c-JUN-mediated LTR transactivation. Our findings demonstrate the importance of the retinoic acid signaling pathway and of cross-coupling interactions in the repression of HIV-1 LTR gene expression.

Brain↗

PubMed1996

SNAP-25 is differentially expressed by noradrenergic and adrenergic chromaffin cells.

This study examines chromaffin cell expression of the synaptosomal-associated protein SNAP-25 in the adrenal medulla by immunoblotting, immunocytochemistry and PCR. Both mRNAs coding for the SNAP-25 isoforms a and b were detected and SNAP-25 was found to be present in all chromaffin cells in adult rat adrenal gland sections. It was essentially restricted to a zone close to the cytoplasmic face of the plasma membrane in the majority of cells, but located extensively throughout the cytoplasm in a chromaffin cell sub-population, identified by double immunofluorescence labelling to have a noradrenergic phenotype. This differential SNAP-25 expression may reflect different stages in the phenotypic development of the sympathoadrenal lineage and be related to an additional functional role in noradrenergic chromaffin cells not associated with secretion.

Adrenal Medulla↗

PubMed1996

Antibacterial activity of secretolytin, a chromogranin B-derived peptide (614-626), is correlated with peptide structure.

Amongst the chromogranin B (CGB) derived fragments naturally generated in bovine chromaffin granules and detected in the extracellular space, we recently identified a major peptide corresponding to the 614-626 sequence of CGB. This peptide, named secretolytin, shared an interesting sequence homology with the lytic domain of cecropins and displayed a potent antibacterial activity. The aim of the present study was to determine the structural features of secretolytin necessary for this biological activity. Our results suggest that an alpha-helical amphipathic structure common to secretolytin, cecropins and pig myeloid antibacterial peptide may account for the antibacterial activity.

Animals↗

PubMed1996

Distinct positive and negative regulatory elements control neuronal and hepatic transcription of the human transferrin gene.

Transferrin (Tf), the iron-transport protein, plays an essential role in the central nervous system development, plasticity, and aging. As a first step toward elucidating the role of each transcription factor involved in the regulation of Tf gene expression, we have recently shown that similar promoter elements direct cell-type specific transcription in oligodendrocytes, epithelial choroid plexus cells, and in the neuronal cell line B103. Here we have analyzed the regulatory elements that control the level of expression of the Tf gene in neuronal cells. Transient expression experiments in B103 cells revealed that the -164/+1 promoter region is stimulated by a position-dependent -1140/-1000 upstream region. DNase I footprinting, gel retardation assays, and antibody reactivity data allowed us to characterize the nuclear factors interacting with this region. The upstream region I-binding protein (URI-BP) belongs to the steroid/retinoid receptor family, while URII-BP is a member of the nuclear factor I (NF-I) family. Interestingly, no enhancer nor silencer activity is detected in B103 cells. This contrasts with our findings in hepatoma cells, where the activity of the -125/+1 promoter can be repressed by a -1000/-819 upstream negative-acting region and stimulated by the -3600/-3300 enhancer. We demonstrate that the negative-acting region presents the characteristics of a silencer that interacts with a nuclear protein present in liver and absent in B103 cells. Similarly, B103 cells lack a nuclear protein able to bind to an essential site of the enhancer. This shows that in B103 cells, the inactivity of the silencer and the enhancer regions results from the absence of at least one essential nuclear protein.

Base Sequence↗

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↗

PubMed1996

Chromogranin A triggers a phenotypic transformation and the generation of nitric oxide in brain microglial cells.

Chromogranin A is an ubiquitous 48,000 mol. wt secretory protein stored and released from many neuroendocrine cells and neurons. In human brain, chromogranin A is a common feature of regions that are known to be affected by various neurodegenerative pathologies such as Alzheimer's, Parkinson's and Pick's diseases. Brain degenerative areas are generally infiltrated by activated microglial cells, the resident macrophage cell population within the central nervous system. Here, we report that both recombinant human chromogranin A and chromogranin A purified from bovine chromaffin granules trigger drastic morphological changes in rat microglial cells maintained in culture. Microglial cells exposed to chromogranin A adopted a flattened amoeboid shape and, this change was associated with an accumulation of actin in the subplasmalemmal region, as observed by immunocytochemistry and confocal laser microscopy. In single microglial cells loaded with indo-1, chromogranin A elicited a rapid and transient increase in [Ca2+]i which preceded the reorganization of actin cytoskeleton. The activity of nitric oxide synthase was estimated by measuring the accumulation of nitrite in the culture medium. Both recombinant human chromogranin A and bovine chromogranin A triggered an important accumulation of nitrite comparable to that induced by lipopolysaccharide, a well-known activator of microglia. The effect of chromogranin A was dose dependent, inhibited by N omega-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, and by cycloheximide, an inhibitor of protein synthesis. These findings suggest that chromogranin A induces an activated phenotype of microglia, and thus may have a role in the pathogenesis of neuronal degeneration in the brain.

Actins↗

PubMed1996

Glucocorticoids and nerve growth factor differentially modulate cell adhesion molecule L1 expression in PC12 cells.

The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels approximately 2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10(-7) M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.

Animals↗

PubMed1996

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells.

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.

Adrenal Medulla↗

PubMed1996

Trimeric G proteins control regulated exocytosis in bovine chromaffin cells: sequential involvement of Go associated with secretory granules and Gi3 bound to the plasma membrane.

Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Galphao antibodies and a synthetic peptide corresponding to the carboxyl terminus of Galphao. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Galphai3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.

Adrenal Medulla↗